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CN101528919A - Mutant DNA polymerases and related methods - Google Patents

Mutant DNA polymerases and related methods Download PDF

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CN101528919A
CN101528919A CNA2007800387722A CN200780038772A CN101528919A CN 101528919 A CN101528919 A CN 101528919A CN A2007800387722 A CNA2007800387722 A CN A2007800387722A CN 200780038772 A CN200780038772 A CN 200780038772A CN 101528919 A CN101528919 A CN 101528919A
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dna polymerase
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archaeal dna
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CN101528919B (en
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K·A·鲍尔
E·S·史密斯
D·H·盖尔芬德
E·菲斯
S·苏科
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Roche Diagnostics GmbH
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Abstract

Disclosed are mutant DNA polymerases having improved extension rates relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Description

Mutant DNA polymerases and methods involving
Invention field
The present invention relates to archaeal dna polymerase field and their various application, comprise the primer extension and the amplification of nucleic acid.
Background of invention
Archaeal dna polymerase is responsible for genomicly duplicating and safeguarding that this is the center responsibility of accurate transfer genetic information between the generation.The function of archaeal dna polymerase in cell is as being responsible for DNA synthetic enzyme.They are having the metal activation agent such as Mg 2+Under the situation about existing, with the dna profiling that is replicated or the sequential polymerization deoxyribonucleoside triphosphate of polynucleotide template domination.In vivo, archaeal dna polymerase participates in a series of DNA building-up processes, comprises dna replication dna, DNA reparation, reorganization and gene amplification.During each DNA building-up process, the repetition DNA template once or at most several times produces consistent replica.In contrast, external, dna replication dna can repeat repeatedly, for example during the polymerase chain reaction (referring to the U.S. Patent number 4,683,202 of for example Mullis).
In the original research of polymerase chain reaction (PCR), archaeal dna polymerase is to add (referring to above-mentioned U.S. Patent number 4,683,202) when each circulation beginning of dna replication dna.Afterwards, confirmed can obtain heat-stable DNA polymerase from the bacterium of growth at high temperature, these enzymes only need add once (referring to the U.S. Patent number 4,889,818 of Gelfand and the U.S. Patent number 4,965,188 of Mullis).Under the high temperature that uses during the PCR, these enzymes do not have irreversible inactivation.Therefore, can carry out the recirculation of polymerase chain reaction and need when each synthetic addition process begins, not add fresh enzyme.Archaeal dna polymerase is the heat-stabilised poly synthase particularly, is the key of technology in a large number in the medical diagnosis of recombinant DNA research and disease.Particularly for diagnostic use, target nucleic acid sequence may only be the sub-fraction of suspicious DNA or RNA, and therefore the words that do not increase may be difficult to detect the existence of target nucleic acid sequence.Because archaeal dna polymerase is in biotechnology and importance medically, the dna polymerase mutant body of the primer extension speed that the enzymatic property that generation has to be needed for example improves, reverse transcription efficient or amplification ability is very useful.
The overall folded mode class of polysaccharase comprises the subdomain of palm, three uniquenesses of finger and thumb like people's the right hand.(referring to Beese etc., Science 260:352-355,1993); Patel etc., BioChemistry 34:5351-5363,1995).The structural changes of finger and thumb subdomain is very big between the different polysaccharase of cell function of size, and the palm subdomain of catalytic is eclipsed all.For example, and the dNTP that introduces interacts and be eclipsed at chemical catalysis is stablized transition state between action period motif A, and the average deviation between the pol of Mammals pol α and protokaryon I family archaeal dna polymerase is approximately 1 (Wang etc., Cell 89:1087-1099,1997).Structurally motif A is extended to alpha-helix mainly to comprise the antiparallel β start of chain of hydrophobic residue.The main aminoacid sequence in dna polymerase activity site is conservative especially.In the example of motif A, for example, sequence D YSQIELR is retained in from the organism of evolving in millions of years for example comprises thermus aquaticus (Thermus aquaticus), chlamydia trachomatis (Chlamydiatrachomatis) and the isolating polysaccharase of intestinal bacteria (Escherichia coli).These observationss are linked together, and they point out that polysaccharase is by similar catalytic mechanism functionating.
Except high conservative, it is relatively variable that the avtive spot of archaeal dna polymerase also is proved to be, and can accept specific aminoacid replacement and significantly do not reduce dna polymerase activity.(referring to the U.S. Patent number 6,602,695 of for example Patel etc.).Such mutant DNA polymerases can provide various selective advantages in diagnosis that for example comprises the nucleic acid building-up reactions and research application.Therefore, need identify amino acid sites in this area, described amino acid sites is suitable for suddenling change to produce the polymerase activity of improvement, comprises extension speed, reverse transcription efficient or the amplification ability of for example improvement.The present invention as set forth herein, satisfies these and other needs.
Summary of the invention
The invention provides the mutant DNA polymerases that the improvement enzymic activity is arranged with respect to corresponding not modified polysaccharase, it can be used for, and multiple nucleic acid is synthetic to be used.In some embodiments, polysaccharase comprises the aminoacid sequence with at least one following motif in the Polymerase Structure territory:
A) X A1-X A2-X A3-X A4-R-X A6-X A7-X A8-K-L-X A11-X A12-T-Y-X A15-X A16(SEQID NO:1); X wherein A1Be I or L; X A2Be L or Q; X A3Be Q, H or E; X A4Be Y, H or F; X A6Be E, Q or K; X A7Be I, L or Y; X A8Be the amino acid except that Q, T, M, G or L; X A11Be K or Q; X A12Be S or N; X A15Be I or V; X A16Be E or D;
B) T-G-R-L-S-S-X B7-X B8-P-N-L-Q-N (SEQ ID NO:2); X wherein B7Be S or T; X B8Be the amino acid except that D, E or N; With
C) X C1-X C2-X C3-X C4-X C5-X C6-X C7-D-Y-S-Q-I-E-L-R (SEQ ID NO:3); X wherein C1Be G, N or D; X C2Be W or H; X C3Be W, A, L or V; X C4Be the amino acid except that I or L; X C5Be V, F or L; X C6Be the amino acid except that S, A, V or G; And X C7Be A or L,
Wherein polysaccharase is with respect to X A8Be the amino acid that is selected from Q, T, M, G or L; X B8Be amino acid and/or the X that is selected from D, E or N C6Be to be selected from the amino acid of S, A, V or G and the identical polysaccharase (promptly with reference to polysaccharase) of others has the reverse transcription efficient that improved nucleic acids acids is extended speed and/or improvement.In some embodiments of reference polysaccharase (for example Z05 or CS5/CS6), X A8Be Q, T, M, G or L, X B8Be D, E or N, X C4Be I or L, and X C6Be S, A, V or G (SEQ ID NOS:23 and 24).In some embodiments of reference polysaccharase, X B8Be D, E or N (SEQ ID NOS:25 and 26).
For a) X of motif A1-X A2-X A3-X A4-R-X A6-X A7-X A8-K-L-X A11-X A12-T-Y-X A15-X A16(SEQ ID NO:1), in some embodiments, X A8Be D-or the L-amino acid that is selected from the group of forming by A, C, D, E, F, H, I, K, N, P, R, S, V, W, Y (SEQ ID NO:27) and analogue thereof.In some embodiments, X A8Be selected from the amino acid (SEQ ID NO:28) of the group of forming by R, K and N.In some embodiments, X A8Be arginine (R) (SEQ ID NO:29).
For motif b) T-G-R-L-S-S-X B7-X B8-P-N-L-Q-N (SEQ ID NO:2), in some embodiments, X B8Be D-or the L-amino acid that is selected from the group of forming by A, C, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (SEQ ID NO:30) and analogue thereof.In some embodiments, X B8Be the amino acid (SEQ ID NO:31) that is selected from the group of forming by G, A, S, T, R, K, Q, L, V and I.In some embodiments, X B8Be the amino acid (SEQ ID NO:32) that is selected from the group of forming by G, T, R, K and L.
For motif c) X C1-X C2-X C3-X C4-X C5-X C6-X C7-D-Y-S-Q-I-E-L-R (SEQ IDNO:3), in some embodiments, X C4Be D-or the L-amino acid that is selected from the group of forming by A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T, V, W, Y (SEQ ID NO:33) and analogue thereof.In some embodiments, X C4Be the amino acid (SEQ ID NO:34) that is selected from the group of forming by F and Y.In some embodiments, X C4Be phenylalanine (F) (SEQID NO:35).In some embodiments, X C6Be the amino acid (SEQ ID NO:36) that is selected from the group of forming by C, D, E, F, H, I, K, L, M, N, P, Q, R, T, W and Y.In some embodiments, X C6Be the amino acid (SEQ IDNO:37) that is selected from the group of forming by F and Y.In some embodiments, X C6Be phenylalanine (F) (SEQ ID NO:38).
In some embodiments, the polysaccharase of improvement (for example Z05 or CS5/CS6) comprises X A8The arginine of position (R), X B8The glycine of position (G), X C4Phenylalanine of position (F) and/or X C6In the phenylalanine of position (F) at least one (SEQ ID NOS:39-68).
In some embodiments, archaeal dna polymerase of the present invention is the modified forms of not modified polysaccharase.In its not modified form, polysaccharase comprises the aminoacid sequence with following motif in the Polymerase Structure territory:
X A1-X A2-X A3-X A4-R-X A6-X A7-X A8-K-L-X A11-X A12-T-Y-X A15-X A16(SEQ IDNO:69); X wherein A1Be I or L; X A2Be L or Q; X A3Be Q, H or E; X A4Be Y, H or F; X A6Be E, Q or K; X A7Be I, L or Y; X A8Be Q, T, M, G or L; X A11Be K or Q; X A12Be S or N; X A15Be I or V; And X A16Be E or D;
T-G-R-L-S-S-X B7-X B8-P-N-L-Q-N (SEQ ID NO:70); X wherein B7Be S or T; And X B8Be D, E or N; With
X C1-X C2-X C3-X C4-X C5-X C6-X C7-D-Y-S-Q-I-E-L-R (SEQ ID NO:71); X wherein C1Be G, N or D; X C2Be W or H; X C3Be W, A, L or V; X C4Be I or L; X C5Be V, F or L; X C6Be S, A, V or G; And X C7Be A or L.
Multiple archaeal dna polymerase is suitable for according to sudden change of the present invention.Especially suitable is the heat-stabilised poly synthase, comprise wild-type or naturally occurring heat-stabilised poly synthase from various thermophile bacteria (thermophilic bacteria), and the heat-stabilised poly synthase that derives from above-mentioned wild-type or naturally occurring enzyme by aminoacid replacement, insertion or disappearance or other modification.Exemplary polysaccharase comprise for example CS5, CS6 or Z05DNA polysaccharase without modified forms, or have the functional DNA polysaccharase of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.Other not modified polysaccharase comprises the archaeal dna polymerase that for example comes from following any kind of thermophile bacteria (or the functional DNA polysaccharase that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with described polysaccharase): Thermotoga maritima (Thermotoga maritima); Thermus aquaticus (Thermus aquaticus); Thermus thermophilus (Thermus thermophilus); The Huang hot bacterium (Thermus flavus) that dwells; The thread hot bacterium that dwells (Thermus filiformis); The hot bacterial classification sps17 (Thermus sp.sps17) of dwelling; The hot bacterial classification Z05 (Thermus sp.Z05) of dwelling; Naples thermobacillus (Thermotoga neopolitana) of dwelling; Hot chamber bacterium (Thermosiphoafricanus) dwells in Africa; Thermus caldophilus or hot hard genus bacillus (Bacillus caldotenax). suitable polysaccharase also comprise those have reversed transcriptive enzyme (RT) active and/or can mix unconventional Nucleotide such as ribonucleotide or other 2 '-polysaccharase of the Nucleotide modified.
In some embodiments, the not modified form of polysaccharase comprises chimeric polysaccharase.In one embodiment, for example, the not modified form of chimeric polysaccharase is CS5 archaeal dna polymerase (SEQ ID NO:18), CS6 archaeal dna polymerase (SEQ ID NO:19) or the polysaccharase that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with CS5 archaeal dna polymerase or CS6 archaeal dna polymerase.In some specific mutation, the not modified form of chimeric polysaccharase comprises one or more aminoacid replacement that is selected from G46E, L329A and E678G with respect to SEQ ID NO:18 or SEQ ID NO:19.For example, the not modified form of mutant polysaccharase can be G46E CS5; G46E L329A CS5; G46EE678G CS5; Or G46E L329A E678G CS5.In exemplary embodiment, replace not modified form so that the mutant polysaccharase of the aminoacid replacement that comprises that one or more is selected from S671F, D640G, Q601R and I669F to be provided.For example, described mutant DNA polymerases can be following any one: G46E S671F CS5; G46E D640G CS5; G46EQ601R CS5; G46E I669F CS5; G46E D640G S671F CS5; G46E L329AS671F CS5; G46E L329A D640G CS5; G46E L329A Q601R CS5; G46EL329A I669F CS5; G46E L329A D640G S671F CS5; G46E S671F E678GCS5; G46E D640G E678G CS5; G46E Q601R E678G CS5; G46E I669FE678G CS5; G46E L329A S671F E678G CS5; G46E L329A D640GE678G CS5; G46E L329A Q601R E678G CS5; G46E L329A Q601RD640G I669F S671F E678G CS5; G46E L329A I669F E678G CS5 etc.
In some embodiments, polysaccharase is CS5 polysaccharase (SEQ ID NO:15), CS6 polysaccharase (SEQ ID NO:16) or Z05 polysaccharase (SEQ ID NO:6), and wherein Xb8 is the amino acid that is selected from the group of being made up of G, T, R, K and L.For example, CS5 or CS6 polysaccharase can be selected from following: D640G, D640T, D640R, D640K and D640L.The Z05 polysaccharase can be selected from the group of being made up of D580G, D580T, D580R, D580K and D580L.
The mutant polysaccharase can comprise other non-replacement modification.A kind of such modification is hot reversible covalent modification, and it makes enzyme deactivation, but at high temperature, such as hatching under the temperature that is generally used for primer extension enzyme is activated.In one embodiment, the mutant polysaccharase that comprises hot reversible covalent modification by heat-stable DNA polymerase and the mixture of dicarboxylic anhydride with one of following formula I or II at alkaline pH be lower than the reaction of carrying out under about 25 ℃ temperature and produce:
Figure A20078003877200101
R wherein 1And R 2Be hydrogen or organic group, they can connect together; Or
Figure A20078003877200111
R wherein 1And R 2Be organic group, they can connect together, and hydrogen is cis.In a specific mutation of above-mentioned enzyme, the not modified form of polysaccharase is G64E CS5.
In some embodiments, as described herein, use single stranded DNA as template (M13mp18 for example, HIV), cause with suitable primer (for example nucleotide sequence 5 '-polynucleotide of GGGAAGGGCGATCGGTGCGGGCCTCTTCGC-3 ' (SEQ ID NO:72)), by measuring the formation that detects double-stranded DNA of mixing, determine to extend speed with this at Fixed Time Interval (for example per 5,10,15,20,30 or 60 seconds) fluorophor.As described herein, during preset time unit, the extension speed of polysaccharase of the present invention can be compared with the extension speed of reference polysaccharase (for example naturally occurring or not modified polysaccharase).
In other each side, the invention provides the recombinant nucleic acid of coding mutant DNA polymerases as herein described, comprise the carrier of this recombinant nucleic acid, and with described carrier transformed host cells.In specific embodiment, described carrier is an expression vector.The host cell that comprises above-mentioned expression vector can be used in of the present invention by cultivate the method for host cell production mutant polysaccharase under the condition that is suitable for express recombinant nucleic acid.Polysaccharase of the present invention can be included in reaction mixture and/or the test kit.The embodiment of recombinant nucleic acid, host cell, carrier, expression vector, reaction mixture and test kit such as preamble and described herein.
On the other hand, provide the method that instructs primer extension.This method is included in substantially under the condition that is suitable for primer extension mutant DNA polymerases of the present invention is contacted with primer, polynucleotide template and free nucleotide, thereby produces the primer that extends.Polynucleotide template can be for example RNA or dna profiling.Free nucleotide can comprise the Nucleotide of unconventional Nucleotide such as ribonucleotide and/or mark.In addition, primer and/or template can comprise one or more nucleotide analog.In some mutation, primer extension method is the method that is used for polynucleotide amplification, is included under the condition that is suitable for polynucleotide amplification mutant DNA polymerases and primer are contacted, polynucleotide template and free nucleotide.
The present invention also provides the test kit that is used for above-mentioned primer extension method.Substantially, described test kit comprises the container that at least one provides mutant DNA polymerases as herein described.In specific embodiment, this test kit further comprises provides one or more to plant one or more other container of other reagent.For example, in specific mutation, one or more other container provides free nucleotide, is suitable for the buffer reagent of primer extension, and/or under the primer extension condition can with the primer of predetermined polynucleotide template hybridization.
The reaction mixture that comprises polysaccharase of the present invention further is provided.Described reaction mixture also can comprise template nucleic acid (DNA and/or RNA), one or more plant primer or probe polynucleotide, free nucleotide (comprising for example Nucleotide, the unconventional Nucleotide of deoxyribonucleotide, ribonucleotide, mark), buffer reagent, salt, marker (for example fluorophor).
Definition
Unless other definition is arranged, all technology used herein and scientific terminology have the implication of understanding usually as the general technical staff of the technical field of the invention.Although only described exemplary method and material, to this paper similar any method is described in fact and raw material all can be used to practice of the present invention or test.For purposes of the invention, following term is defined as follows.
Term " one " and " being somebody's turn to do " comprise plural object, unless other implication clearly pointed out in context.
" amino acid " refers to and can merge into peptide, polypeptide or proteinic any monomer unit.As described herein, term " amino acid " comprises the a-amino acid of following 20 kinds of natural or genetic codings: L-Ala (Ala or A), arginine (Arg or R), l-asparagine (Asn or N), aspartic acid (Asp or D), halfcystine (Cys or C), glutamine (Gln or Q), L-glutamic acid (Glu or E), glycine (Gly or G), Histidine (His or H), Isoleucine (Ile or I), leucine (Leu or L), Methionin (Lys or K), methionine(Met) (Met or M), phenylalanine (Phe or F), proline(Pro) (Pro or P), Serine (Ser or S), Threonine (Thr or T), tryptophane (Trp or W), tyrosine (Tyr or Y) and Xie Ansuan (Val or V).The structure of these 20 kinds of natural amino acids is seen for example Stryer etc., BioChemistry, 5 ThEd., Freeman and Company (2002).Other amino acid such as seleno-cysteine and pyrroles's Methionin also can genetic coding (Stadtman (1996) " Selenocysteine, " Annu Rev Biochem.(2002) " Genetic code:introducingpyrrolysine, " such as 65:83-100 and Ibba Curr Biol.12 (13): R464-R466).Term " amino acid " also comprises the amino acid (for example side chain and/or main chain are modified) and the amino acid analogue of alpha-non-natural amino acid, modification.Referring to (2004) " Selective incorporation of 5-hydroxytryptophaninto proteins in mammalian cells, " such as for example Zhang ProC.Natl.Acad.Sci.U.S.A.101 (24): 8882-8887, Anderson etc. (2004) " An expanded genetic code with afunctional quadruplet codon " ProC.Natl.Acad.Sci.U.S.A.101 (20): 7566-7571, Ikeda etc. (2003) " Synthesis of a novel histidineanalogue and its efficient incorporation into a protein in vivo, " Protein Eng. Des.Sel.16 (9): 699-706, Chin etc. (2003) " An Expanded Eukaryotic GeneticCode, " Science301 (5635): 964-967, James etc. (2001) " Kineticcharacterization of ribonuclease S mutants containing photoisomerizablephenylazophenylalanine residues, " Protein Eng.Des.Sel.14 (12): 983-991, Kohrer etc. (2001) " Import of amber and ochre suppressor tRNAs intomammalian cells:A general approach to site-specific insertion of aminoacid analogues into proteins, " ProC.Natl.Acad.Sci.U.S.A.98 (25): 14310-14315, Bacher etc. (2001) " Selection and Characterization ofEscherichia coli Variants Capable of Growth on an Otherwise ToxicTryptophan Analogue, " J.Bacteriol.183 (18): 5414-5425, Hamano-Takaku etc. (2000) " A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes theUnnatural Amino Acid Azatyrosine More Efficiently than Tyrosine, " J.Biol. Chem.275 (51): (2001) " Proteins with{beta}-(thienopyrrolyl) alanines as alternative chromophores andpharmaceutically active amino acids, " such as 40324-40328 and Budisa Protein Sci.10 (7): 1281-1292.
Further specify, amino acid typically is that comprise replacement or unsubstituted amino, replacement or unsubstituted carboxyl and one or more are planted side chain or group, or any one the organic acid of analogue of these groups.Exemplary side chain comprises, for example the arbitrary combination of sulfydryl, seleno, alkylsulfonyl, alkyl, aryl, acyl group, ketone group, azido-, hydroxyl, hydrazine, cyano group, halogen, hydrazides, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocycle, ketenes, imines, aldehyde, ester, thioic acid sulfoacid, azanol or these groups.Other representational amino acid includes but not limited to, comprises the amino acid of photosensitive crosslinker, melts combine amino acid, the amino acid of spin labeling, fluorescigenic amino acid, the amino acid that comprises metal, the amino acid that contains new functional group, with the covalently or non-covalently interactional amino acid of other molecule, to light shakiness (photocaged) but and/or the amino acid of photoisomerization, radioactivity amino acid, the amino acid that comprises vitamin H or vitamin H analogue, glycosylation amino acid, the amino acid of other carbohydrate modification, the amino acid that comprises polyoxyethylene glycol or polyethers, the amino acid that heavy atom replaces, chemistry amino acid that can split and/or that light can split, comprise the amino acid that carbon connects sugar, redox active amino acids, comprise the amino acid of amino thioic acid sulfoacid and comprise one or more toxicity amino acid partly.
The expression of term " mutant " in archaeal dna polymerase context of the present invention comprises the polypeptide of one or more aminoacid replacement with respect to the function corresponding archaeal dna polymerase, is typically reorganization.
Term in mutant polysaccharase context " not modified form " is the term that is used for defining mutant DNA polymerases of the present invention in this article: term " not modified form " refers to have the functional DNA polysaccharase of the mutant polysaccharase aminoacid sequence except that one or more amino acid sites of specific conduct sign mutant polysaccharase.Therefore, with (a) its not modified form and (b) one or more specific amino acids replace the mutant DNA polymerases of mentioning represent that except that specific aminoacid replacement the mutant polysaccharase has and not modified identic other aminoacid sequence in specific motif.Polysaccharase can comprise other sudden change so that want functional to be provided, for example the Nucleotide of Gai Liang bi-deoxyribose Nucleotide, ribonucleotide, ribonucleoside acid-like substance, dye marker mixes, 5 of adjustment '-nuclease, 3 of adjustment '-nuclease (or correction) activity etc.In view of the above, implementing as herein describedly when of the present invention, the not modified form of archaeal dna polymerase is predetermined.The not modified form of archaeal dna polymerase can be, for example wild-type and/or naturally occurring archaeal dna polymerase, or the archaeal dna polymerase of modified consciously.The preferred heat-stable DNA polymerase of form that polysaccharase is not modified is for example from the archaeal dna polymerase of various thermophile bacteria and the functional variant that has sequence identity basically with wild-type or naturally occurring heat-stabilised poly synthase.Above-mentioned variant can comprise, chimeric dna polymerase for example, and such as U.S. Patent number 6,228,628 and the chimeric dna polymerase described of U. S. application publication number 2004/0005599.In specific embodiment, the not modified form of described polysaccharase has reversed transcriptive enzyme (RT) activity.
Term " heat-stabilised poly synthase " refers to heat stable enzyme, and it is heat-stable, and after at high temperature passing through the needed time of double-strandednucleic acid sex change, it can keep enough activity carrying out primer extension reaction subsequently, and does not have irreversible denaturation (inactivation).The required heating condition of nucleic acid denaturation is well known in the art and in for example U.S. Patent number 4,683,202,4,683,195 and 4,965,188 illustrated.As described herein, the heat-stabilised poly synthase is suitable for using in reaction such as the polymerase chain reaction (" PCR ") that temperature cycle changes.Irreversible denaturation herein refers to permanent and loss of enzyme activity completely.For the heat-stabilised poly synthase, enzymic activity refers to make up by rights Nucleotide with the katalysis of formation with template nucleic acid chain complementary primer extension product.Heat-stable DNA polymerase from thermophile bacteria comprises, for example from Thermotoga maritima, thermus aquaticus, thermus thermophilus, the Huang hot bacterium that dwells, the thread hot bacterium that dwells, the hot bacterial classification sps17 of dwelling, the hot bacterial classification Z05 of dwelling, Thermuscaldophilus, the hard genus bacillus of heat, Naples the dwell archaeal dna polymerase of hot chamber bacterium of thermobacillus and Africa of dwelling.
As used herein, " chimeric " protein refers to the protein of aminoacid sequence representative from the fusion product of the subsequence of at least two different protein amino acid sequences.The chimeric protein typical case is not the direct control generation by aminoacid sequence, but comes out from " chimeric " genetic expression of this chimeric aminoacid sequence of encoding.In specific embodiment, for example, mutant DNA polymerases of the present invention is the chimeric protein that origin aminoterminal (N-end) zone that comes from Thermus bacterial classification archaeal dna polymerase and carboxyl terminal (C-end) zone that derives from the Tma archaeal dna polymerase are formed without modified forms.The N-stub area refers to extend to from N-end (amino acid position 1) zone of internal amino acid.Similarly, the C-stub area refers to that amino acid internally extends to the zone of C-end.
In the context of mutant DNA polymerases, be based on according to the numbering convention of Nucleotide or amino acid whose positional number and with the mode aligned sequences of maximization sequence identity per-cent with " corresponding " of another sequence (for example zone, fragment, Nucleotide or amino acid position etc.).Because it is identical not to be that given " respective regions " inner all sites all needs, the non-coupling site of respective regions inside may be considered to " corresponding site ".In view of the above, as used herein, " with the corresponding amino acid sites of amino acid sites [X] " of specific archaeal dna polymerase represents that other is through the archaeal dna polymerase of checking and the set in the equivalent site in structure homologue and the family.In typical embodiment of the present invention, " corresponding " of amino acid sites determined according to the polysaccharase zone of the motif that comprises one or more SEQ ID NO:1, SEQ ID NO:2 that further inquires into and SEQ ID NO:3 herein.
" recombinant chou " used herein refers to pass through the recombination method aminoacid sequence or the nucleotide sequence of modified consciously.Term herein " recombinant nucleic acid " expression is operated via the nucleic acid of endonuclease usually, the initial nucleic acid that is different from the conventionally form in the nature in external generation.Therefore the isolating mutant DNA polymerases nucleic acid of linear forms or all be considered to recombinant chou of the present invention by the expression vector that connects the external generation of common uncombined dna molecular.Be clear that very much, in case recombinant nucleic acid is manufactured and introduce host cell again, it with non-reorganization duplicate, that is, use intravital cell mechanism of host cell rather than manipulation in vitro; Yet above-mentioned nucleic acid in a single day reorganization produces, although non-subsequently recombinant replication still is considered to recombinant chou of the present invention." recombinant protein " is to use recombinant technology, i.e. the protein of making by the expression of above-mentioned recombinant nucleic acid.The recombinant protein typical case is according at least one or more a plurality of feature and be different from naturally occurring protein.
Nucleic acid is " can handle and be connected " when having functional relationship with another nucleotide sequence.For example, if promotor or enhanser influence transcribing of encoding sequence, it can be handled with encoding sequence and be connected so; If perhaps ribosome bind site is positioned to promote translation, it can be handled with encoding sequence and be connected so.
Term " host cell " refers to the one cell from higher plant or animal of growing in single celled prokaryotic organism and eukaryote organism (for example bacterium, yeast and actinomycetes) and the cell culture.
Term " carrier " refers to a DNA, and the typical case is double-stranded, and it can be to have inserted a foreign DNA.Described carrier can be for example to derive from plasmid.Carrier is included in " replicon " polynucleotide sequence that promotes self-replicating in the host cell.Foreign DNA is defined as allogenic DNA, and it is not the DNA of natural discovery in this host cell, and for example, its replicating vector molecule, coding can be selected the mark that maybe can screen, the transgenosis of perhaps encoding.Carrier is used to transport external or allogenic DNA enters proper host cell.In case in host cell, carrier can be independent of that host chromosome DNA duplicates or duplicate simultaneously with host chromosome DNA, can produce some copies of the DNA of carrier and insertion thereof.In addition, can also to comprise that the DNA that allows insertion is transcribed into the mRNA molecule or makes the dna replication dna of insertion on the contrary be the necessary element of multiple copied RNA to carrier.Some expression vector also comprises near the sequential element of DNA of insertion, and it can increase the transformation period of the mRNA of expression, and/or allows described mRNA to translate into protein molecular.Therefore the many mRNA and the peptide molecule of the dna encoding that inserts can promptly synthesize.
Term " Nucleotide " is except that referring to naturally occurring ribonucleotide or deoxyribonucleotide monomer, it should also be understood that herein to relating to its relevant structural variant, comprise derivative and analogue, it for the specific context that uses this Nucleotide (for example, with complementary base hybridization) be equivalent on function, unless context spells out difference.
Term " nucleic acid " or " polynucleotide " refer to polymer, and it can be corresponding to Yeast Nucleic Acid (RNA) or thymus nucleic acid (DNA) polymer or its analogue.It comprises the polymer of Nucleotide such as RNA and DNA, with and synthesized form, modification (for example chemistry or biological chemistry are modified) form and mixed polymerization body (for example comprising RNA and DNA subunit).Exemplary modification comprises and methylating, replace one or more naturally occurring Nucleotide with analogue, modification between Nucleotide such as uncharged key (for example methyl-phosphonate, phosphotriester, phosphoamide thing, carbaminate etc.), lateral parts (for example polypeptide), intercalator (for example acridine, psoralene etc.), sequestrant, alkylating agent and the key modified (for example different nucleic acid of α etc.).Also comprise the synthetic molecules of simulation polynucleotide by hydrogen bond and other chemical interactions and specified sequence bonded ability.Though the nucleic acid of synthesized form can comprise that (for example, ((peptide nucleic acid(PNA) that (Science254:1497-1500,1991) are described), typically nucleotide monomer connects by phosphodiester bond Nielsen etc. other key.Nucleic acid can be or comprise, for example, and product, oligonucleotide, probe and the primer of karyomit(e) or chromosome segment, carrier (for example expression vector), expression cassette, naked DNA or RNA polymer, polymerase chain reaction (PCR).Nucleic acid can be, for example, and strand, two strands or three chains, and be not limited to any particular length.Unless otherwise noted, except that any sequence that spells out, specific nucleotide sequence randomly comprises or the complementary sequence of encoding.
Term " oligonucleotide " refers to comprise the nucleic acid of at least 2 nucleic acid monomer units (for example Nucleotide).Oligonucleotide typically comprises about 6 to about 175 nucleic acid monomer units, more typically about 8 to about 100 nucleic acid monomer units, more typically about 10 to about 50 nucleic acid monomer units (for example about 15, about 20, about 25, about 30, about 35, or more nucleic acid monomer unit).The definite size of oligonucleotide depends on many factors, comprises the final function or the purposes of this oligonucleotide.Oligonucleotide at random passes through prepared by any suitable process, include but not limited to the separation of existing or native sequences, dna replication dna or amplification, reverse transcription, the clone and the restrictive diges-tion of suitable sequence, or for example pass through, phosphotriester method (the Meth.Enzymol.68:90-99 of Narang etc., 1979), phosphodiester method (the Meth.Enzymol.68:109-151 of Brown etc., 1979), the diethyl phosphoramidite method of Beaucage etc. (Tetrahedron Lett.22:1859-1862,1981), the triester method (J.Am.Chem.Soc.103:3185-3191 of Matteucci etc., 1981), automatically synthesis method, or authorized the U.S. Patent number 4,458 of Caruthers etc. " PROCESS FORPREPARING POLYNUCLEOTIDES " by name on July 3rd, 1984,066 solid phase Zhi Chifa, or the method direct chemical of other method known to those skilled in the art is synthetic.
Term used herein " primer " refers to can be as the polynucleotide in the nucleic acid synthetic site of starting template guidance (for example, be included in the suitable damping fluid and exist under suitable temperature or the temperature cycle (for example in the polymerase chain reaction) under the condition of required nucleoside triphosphate (under the template domination that will duplicate) and polysaccharase) under the condition that initial primers is extended.For further specifying, primer can also be used for other multiple oligonucleotide mediated building-up process, comprises the initiator as the RNA de novo synthesis process relevant with in-vitro transcription (for example based on the amplification (NASBA) of nucleotide sequence, the amplification (TMA) of transcriptive intermediate etc.).The primer typical case is single stranded oligonucleotide (a for example oligodeoxyribonucleotide).The suitable length of primer depends on the purposes that this primer is predetermined, but typically from 6 to 40 Nucleotide, more typically from 15 to 35 Nucleotide.Short primer molecule needs lower temperature to form sufficiently stable hybridization complex with template usually.Primer does not need to reflect the accurate sequence of template, but must be that enough complementary to hybridize with template primer extension take place.In specific embodiment, one group of primer of term " primer to " expression, it comprises with 5 of the complementary sequence hybridization of 5 of the nucleotide sequence of amplification ' terminal ' have adopted primer (being sometimes referred to as " forward ") and with 3 ' antisense primer of 3 ' terminal hybridization of the sequence of amplification and (is sometimes referred to as " oppositely ") (for example, if target sequence as rna expression or RNA).If desired, primer can by with combine mark with the detectable mark of spectroscopy, photochemical, biochemical, immunochemical or chemical method.For example, useful mark comprises 32P, fluorescence dye, electron dense reagent, enzyme (being generally used for enzyme linked immunological absorption (ELISA) determination test), vitamin H or haptens and protein can utilize antiserum(antisera) or monoclonal antibody for it.
When relating to nucleic acid base, nucleoside triphosphate or Nucleotide, in the polynucleotide that term " routine " or " natural " refer to describe naturally occurring (that is being dATP, dGTP, dCTP and dTTP) for DNA.In addition, external synthesis reaction of DNA such as order-checking in, dITP and 7-denitrogenation-dGTP often are used to replace dGTP, and 7-denitrogenation-dATP can be used for replacing dATP.Jointly, these are called dNTPs.
When relating to nucleic acid base, nucleosides or Nucleotide, term " unconventional " or " modification " are included in modifier, derivative or the analogue of base, nucleosides or the Nucleotide of naturally occurring routine in the specific polynucleotide.Compare with the dNTPs of routine, specific unconventional Nucleotide is modified in 2 ' position of ribose.Therefore, although for the naturally occurring Nucleotide of RNA is that ribonucleotide (is ATP, GTP, CTP, UTP, nominal rNTPs), because these Nucleotide have the hydroxyl in sugar 2 ' position, dNTPs does not have by comparison, as used herein, the ribonucleotide as the substrate of archaeal dna polymerase is unconventional Nucleotide.As used herein, unconventional Nucleotide includes but not limited to be used as the compound of terminator in nucleic acid sequencing.Exemplary terminator compound includes but not limited to have 2 ', the compound that is called dideoxyribonucleoside triphosphate of 3 ' two deoxidation structures.Dideoxyribonucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP jointly are called ddNTPs.Other examples of terminator compound comprise 2 of ribonucleotide '-PO 4Analogue (referring to, U. S. application publication number 2005/0037991 and 2005/0037398 for example).Other unconventional Nucleotide comprises thiophosphoric acid dNTPs ([[α]-S] dNTPs), 5 '-[α]-borine (borano)-dNTPs, [α]-methyl-phosphonate dNTPs and ribonucleotide triphosphate (rNTPs).Unconventional base can with radio isotope such as 32P, 33P or 35S, fluorescent mark, chemiluminescent labeling, bioluminescence marker, hapten-marked such as vitamin H, perhaps enzyme labelling such as Streptavidin or avidin come mark.Fluorescent mark can comprise electronegative dyestuff, the dyestuff of fluorescein family for example, perhaps electroneutral dyestuff, the dyestuff of rhodamine family for example, perhaps positively charged dyestuff, for example dyestuff of Hua Jing family.The dyestuff of fluorescein family comprises, for example FAM, HEX, TET, JOE, NAN and ZOE.The dyestuff of rhodamine family comprises texas Red, ROX, R110, R6G and TAMRA.Perkin-Elmer (Boston, MA), AppliedBiosystems (Foster City, CA) or Invitrogen/Molecular Probes (Eugene OR) sells various dyestuffs or with the Nucleotide of FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, texas Red and TAMRA mark.The dyestuff of Hua Jing family comprises Cy2, Cy3, Cy5 and Cy7, its GE Healthcare UK Limited (Amersham Place, LittleChalfont, Buckinghamshire, England) on sale.
As used herein, " sequence identity per-cent " is by determining in the sequence of two best comparisons of comparison window comparison, wherein for the best comparison of two sequences, the sequence part in comparison window is compared with reference sequences (do not comprise and adding or disappearance) can comprise interpolation or disappearance (being breach).Per-cent be number by the site of determining to be present in nucleic acid base identical in the two sequences or amino-acid residue to obtain mating the number in site, remove the number in coupling site and take advantage of this result recently to calculate with the sum of the site in the comparison window to obtain sequence identity percentage with 100.
In the context of two or more nucleic acid or peptide sequence, term " identical " or " identity " per-cent refer to when use in comparison window or specified zone a kind of in the following sequence comparison algorithm or by hand comparison and visual inspection be measured as maximal phase should and relatively and during comparison, identical or have the identical Nucleotide of particular percentile or amino-acid residue (for example 60% identity in the specific region, randomly 65%, 70%, 75%, 80%, 85%, 90% or 95% identity) two or more sequences or a subsequence.As infructescence at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, perhaps at least 55% is identical, and they are " substantially the same " so.These definition also refer to the complementary sequence of cycle tests.Randomly, identity is present in the zone at least about 50 length of nucleotides, or more typically 100 to 500 or 1000 or more in the zone of polynucleotide length.
In the context of two or more nucleic acid or peptide sequence, term " similarity " or " similarity per-cent " refer to when use in comparison window or specified zone a kind of in the following sequence comparison algorithm or by hand comparison and visual inspection be measured as maximal phase should and relatively and during comparison, have particular percentile identical or two of being defined as the similar amino-acid residue that conserved amino acid replaces (for example 60% similarity in the specific region, randomly 65%, 70%, 75%, 80%, 85%, 90% or 95% is similar) or many sequences or subsequence.As infructescence at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, perhaps at least 55% is similar each other, and they are " similar basically " each other so.Randomly, similarity is present in the zone at least about 50 amino acid lengths, or more typically about at least 100 to 500 or 1000 or more in the zone of amino acids length.
For sequence relatively, usually with a sequence as the reference sequence, with cycle tests and its comparison.When using sequence comparison algorithm, will test and reference sequences input computer, specify subsequence coordinate (coordinate), if necessary, also need specified sequence algorithm routine parameter.Usually use the default program parameter, perhaps can specify alternative parameter.Based on program parameter, sequence comparison algorithm calculates sequence identity or the similarity per-cent of cycle tests with respect to reference sequences then.
As used herein, " comparison window " comprises and relates to the fragment that is selected from by the continuous site of arbitrary number of 20 to 600 groups of forming, common about 50 to about 200, more generally about 100 to about 150, wherein the best comparison of two sequences back sequence can with the reference sequences in the continuous site of same number relatively.It is well known in the art being used for the sequence alignment method of comparison.It is passable to be used for the optimal sequence comparison of comparison, for example, adopt the algorithm (Adv.Appl.Math.2:482 of local homology of Smith and Waterman, 1970), with the homology alignment algorithm (J.Mol.Biol.48:443,1970) of Needleman and Wunsch, with search similarity method (the Proc.Natl.Acad.Sci.USA 85:2444 of Pearson and Lipman, 1988), (for example, the GAP among the theWisconsin Genetics Software Package, BESTFIT are carried out in computerize with these algorithms, FASTA, and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, Wis.), perhaps comparison and visual inspection by hand (referring to, for example, Ausubel etc., Current Protocols inMolecular Biology (1995 supplement)).
The example of a useful algorithm is PILEUP.PILEUP uses progressive, paired comparison to set up the multiple sequence comparison to show association and sequence identity per-cent from one group of correlated series.Its same drafting tree or tree derivation show bunch mutual relationship that is used to compare.PILEUP uses the simplification progression comparison method (J.Mol.Evol.35:351-360,1987) of Feng and Doolittle.The method (CABIOS 5:151-153,1989) that method of using and Higgins and Sharp describe is similar.This program can be compared the most nearly 300 sequences, and wherein the maximum length of each bar is 5,000 Nucleotide or amino acid.This multiple ratio to program from two sequences the most similar of paired comparison, produce two aligned sequences bunch.Then will this bunch and bunch comparison of the sequence of time maximally related sequence or comparison.Compare two bunches of sequences by the simple extension that two independent sequences are compared in pairs.Final comparison realizes by a series of progressive, paired comparisons.Program by specifying specific sequence and sequence comparison domain thereof amino acid or nucleotide coordinate and move by the designated program parameter.Use PILEUP, use following parameters that reference sequences is compared with other cycle tests to determine that sequence identity concerns per-cent: default gap weight (3.00), default gap length weight (0.10) and weighting stop breach.PILEUP can from GCG sequence analysis software bag, obtain (as, 7.0 editions (Devereaux etc., Nuc.Acids Res.12:387-95,1984) or more late).
Another example that is suitable for the algorithm of definite sequence identity and sequence similarity per-cent is BLAST and BLAST 2.0 algorithms, it is respectively by (Nuc.Acids Res.25:3389-402 such as Altschul, 1977) and Altschul etc. (J.Mol.Biol.215:403-10,1990) describe.Being used to carry out the software that BLAST analyzes can obtain publicly by state-run biotechnology information center (http://www.ncbi.nlm.nih.gov/).This algorithm comprises: at first discern high sub-sequence to (HSPs) by the short word section of identification length W in search sequence, its coupling or satisfy certain positive threshold value scoring T with the same length field comparison of database sequence the time.T is considered to contiguous field scoring threshold value (above-mentioned Altschul etc.).These initial contiguous fields are hit as the seed of initial search to find to comprise their longer HSPs.Field is hit along the two-way extension of each sequence, as long as the comparison mark of accumulative total increases.For nucleotide sequence, cumulative point operation parameter M (the right prize branch of coupling residue; All the time greater than 0) and the N (point penalty of the residue that do not match; All the time less than 0) calculate.For aminoacid sequence, use rating matrix to calculate cumulative point.The extension that field is hit in all directions stops at: accumulative total is compared mark when its maximum acquisition value reduces quantity X; Because one or more negative accumulation that divides the residue comparison, cumulative point reaches or is lower than at 0 o'clock; Perhaps arrive the end of arbitrary sequence.The sensitivity and the speed of BLAST algorithm parameter W, T and X decision comparison.BLASTN program (being used for nucleotide sequence) is used acquiescence word length (W) 11, expected value (E) 10, and M=5, N=-4, and compare two chains.For aminoacid sequence, the BLASTP program is used acquiescence word length 3, expected value (E) 10, with BLOSUM62 rating matrix (referring to Henikoff and Henikoff, ProC.Natl.Acad.Sci.USA 89:10915,1989) comparison (B) 50, expected value (E) 10, M=5, N=-4, and compare two chains.
The BLAST algorithm also carry out similarity between the two sequences statistical study (referring to, for example, Karlin and Altschul, ProC.Natl.Acad.Sci.USA 90:5873-87,1993).A kind of similarity measurement that the BLAST algorithm provides is that (P ((N)), it provides the indication of the occurrent probability of coupling between two Nucleotide or the aminoacid sequence to minimum summation probability.For example, if minimum summation probability is lower than approximately 0.2 in the comparison of test nucleic acid and reference nucleic acid, typically be lower than approximately 0.01, and more typically be lower than approximately 0.001, this nucleic acid is considered to similar to reference sequences so.
Term " nucleic acid extension speed " refers to biological catalyst (enzyme for example, such as polysaccharase, ligase enzyme etc.) with template dependence or non-template dependence mode one or more Nucleotide is added to the speed that (for example covalently) extends this nucleic acid (for example, primer or other oligonucleotide) on the nucleic acid.With respect to the not modified form of archaeal dna polymerase, specific mutant DNA polymerases described herein has improved nucleic acids acids with respect to the unmodified form of these archaeal dna polymerases and extends speed, makes that they can extend primer with the speed higher than not modified form under one group of given reaction conditions.
Term " reverse transcription efficient " refers to that RNA molecule reverse transcription is the mark of cDNA in given reverse transcription reaction.In specific embodiment, with respect to the not modified form of these archaeal dna polymerases, mutant DNA polymerases of the present invention has the reverse transcription efficient of improvement.That is to say that mutant DNA polymerases is than their the not modified form reverse transcription RNA template of balloon score more under one group of specific reaction conditions.
The accompanying drawing summary
Fig. 1 has described the aminoacid sequence comparison from zone in the Polymerase Structure territory of the exemplary heat-stable DNA polymerase of multiple thermophile bacteria: thermus thermophilus (Tth) (SEQ ID NO:4), Thermus caldophilus (Tca) (SEQ ID NO:5), the hot bacterial classification Z05 (Z05) (SEQ IDNO:6) of dwelling, thermus aquaticus (Taq) (SEQ ID NO:7), the Huang hot bacterium (Tfl) (SEQ ID NO:8) that dwells, the thread hot bacterium (Tfi) that dwells (SEQ ID NO:9), the hot bacterial classification sps17 (Sps17) (SEQ ID NO:10) of dwelling, Thermotoga maritima (Tma) (SEQ ID NO:11), Naples thermobacillus (Tne) (SEQ IDNO:12) of dwelling, hot chamber bacterium (Taf) (SEQ ID NO:13) and hot hard genus bacillus (Bca) (SEQID NO:14) dwell in Africa.The aminoacid sequence comparison also comprises from the zone in the Polymerase Structure territory of the representational chimeric heat-stable DNA polymerase of CS5 (SEQ ID NO:15) by name and CS6 (SEQ ID NO:16).In addition, also comprise the sequence (Cons) (SEQ ID NO:17) that is presented at the consistent aminoacid sequence in these exemplary sequence.Further, the polypeptide zone that shows comprises amino acid motif XXXXRXXXKLXXTYXX (SEQ ID NO:1), TGRLSSXXPNLQN (SEQ ID NO:2) and XXXXXXXDYSQIELR (SEQ ID NO:3), and mutable site wherein is further definition herein.These motifs highlight with runic in each polysaccharase sequence.Be suitable for according to the amino acid sites of sudden change of the present invention with asterisk ( *) point out.Breach in the comparison is pointed out with round dot (.).
Fig. 2 A provides the aminoacid sequence (SEQ IDNO:18) of chimeric heat-stable DNA polymerase CS5.
Fig. 2 B provides the nucleotide sequence (SEQ IDNO:20) of the chimeric heat-stable DNA polymerase CS5 of coding.
Fig. 3 A provides the aminoacid sequence (SEQ IDNO:19) of chimeric heat-stable DNA polymerase CS6.
Fig. 3 B provides the nucleotide sequence (SEQ IDNO:21) of the chimeric heat-stable DNA polymerase CS6 of coding.
Fig. 4 is the bar graph that shows the stdn extension speed of the various mutant of G46E L329A E678G (GLE) CS5 archaeal dna polymerase.Speed is extended in the representative of y axle relatively, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G).The extension rate value that the mutant polysaccharase obtains comes stdn with respect to the extension rate value that the GLE CS5 archaeal dna polymerase that is made as 1.00 obtains.
Fig. 5 is the bar graph that shows the stdn extension speed of the various mutant of G46E L329A E678G (GLE) CS5 archaeal dna polymerase.Speed is extended in the representative of y axle relatively, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G).The extension rate value that the mutant polysaccharase obtains comes stdn with respect to the extension rate value that the GLE CS5 archaeal dna polymerase that is made as 1.00 obtains.
Fig. 6 shows the Z05 archaeal dna polymerase, Δ Z05 DNA (dZ05 among Fig. 6) polysaccharase is (referring to the U.S. Patent number 5 of " the MUTATEDTHERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROMTHERMUS SPECIES Z05 " by name that authorize October 3 nineteen ninety-five such as Abramson etc., 455, authorized the U.S. Patent number 5 of Abramson etc. " DNA ENCODING THERMOSTABLENUCLEIC ACID POLYMERASE ENZYME FROM THERMUS SPECIESZ05 " by name on October 7th, 170 and 1997,674,738) and the stdn of the various mutant of G46E L329A (GL) CS5 archaeal dna polymerase extend the bar graph of speed.Speed is extended in the representative of y axle relatively, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G).The extension rate value that the mutant polysaccharase obtains comes stdn with respect to the extension rate value that the GLE CS5 archaeal dna polymerase that is made as 1.00 obtains.
Fig. 7 is the bar graph that shows the stdn extension speed of Z05 archaeal dna polymerase, Δ Z05 DNA (dZ05 among Fig. 7) polysaccharase and the various mutant of G46E L329A (GL) CS5 archaeal dna polymerase.Speed is extended in the representative of y axle relatively, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G).The extension rate value that the mutant polysaccharase obtains comes stdn with respect to the extension rate value that the GLE CS5 archaeal dna polymerase that is made as 1.00 obtains.
Fig. 8 is the chart that different archaeal dna polymerases extend speed under salt (KOAc) concentration that is presented at variation.Speed (arbitrary unit) is extended in the representative of y axle, and the x axle is represented KOAc concentration (mM).The incidental legend of chart shows in the chart archaeal dna polymerase corresponding to each bar trace.Especially, delta Z05 represents Δ Z05 archaeal dna polymerase, and Z05 represents the Z05 archaeal dna polymerase, other enzyme representative of indication has specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G) mutant CS5DNA polysaccharase.
Fig. 9 is the chart that different archaeal dna polymerases extend speed under salt (KOAc) concentration that is presented at variation.Speed (arbitrary unit) is extended in the representative of y axle, and the x axle is represented KOAc concentration (mM).The incidental legend of chart shows in the chart archaeal dna polymerase corresponding to each bar trace.Especially, other enzyme representative of indication has the mutant CS5 archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G).
Figure 10 is the bar graph that is presented at threshold circulation (Ct) value that various mutant CS5 archaeal dna polymerases obtain among the RT-PCRs.The y axle is represented the Ct value, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, and S=S671F).
Figure 11 is the Mg that is presented at the RT incubation time with variation + 2The bar graph of threshold circulation (Ct) value that various mutant CS5 archaeal dna polymerases obtain among the activatory RT-PCRs.The y axle is represented the Ct value, and the representative of x axle has the archaeal dna polymerase of specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, and S=S671F).
Figure 12 is the Mn that is presented at the RT incubation time with variation + 2The bar graph of threshold circulation (Ct) value that various mutant CS5 archaeal dna polymerases obtain among the activatory RT-PCRs.The y axle is represented the Ct value, and the representative of x axle has specified point sudden change (G=G46E, L=L329A, Q=Q601R, D=D640G, archaeal dna polymerase S=S671F).
Figure 13 A and B are explanation some enzyme described herein produces the ability of total length amplicon under the various conditions that comprise ribonucleotide sepharose photos.As mark on the photo, the enzyme of test is GQDSE, CS6-GQDSE, GLQDSE, GDSE, GLDSE, GLDE, GE (G46ECS5R) and 4: 1 blended GL and GLE (GL CS5/GLE), G=G46E wherein, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G.All these enzymes all are the CS5 enzymes except that the enzyme of CS6-GQDSE indication.
Figure 14 A be delta Cts (y axle) about the enzyme of describing among Figure 13 A and the B to the chart of various rATP conditions (y axle) of test, and Figure 14 B to be the rNTP of the enzyme described about Figure 13 A and B mix the chart of percentage (y axle) to the various rNTP conditions (y axle) of test.
Figure 15 A and B are explanation some enzyme described herein produces the ability of total length amplicon under the various conditions that comprise biotinylated ribonucleotide sepharose photos.As mark on the photo, the CS5 enzyme of test is GQDSE, GDSE, GE (G46E CS5R) and 4: 1 blended GL and GLE (GL/GLE mixes (4: 1)), G=G46E wherein, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G.
Figure 16 A is the chart of the delta Cts (y axle) of the enzyme (x axle) described about Figure 15 A and B under the various rCTP conditions (legend) of test, and Figure 14 B is various biotin labeled rCTP condition (legend) chart of the delta Cts (y axle) of those enzymes (x axle) down of test.
Figure 17 is presented at the bar graph of the middle enzyme concn of the pyrophosphorolysis activatory polymerization (PAP) that utilizes G46E L329A E678G (GLE) CS5 archaeal dna polymerase to the effect of threshold circulation (Ct) value.The y axle is represented the Ct value, and the x axle is represented enzyme concn (nM).The incidental legend of chart shows that the copy number corresponding to the template nucleic acid of each bar trace among the figure (does not have template nucleic acid copy (notemp), 1e 4Individual template nucleic acid copy (1E4/rxn), 1e 5Individual template nucleic acid copy (1E5/rxn), 1e 6Individual template nucleic acid copy (1E6/rxn)).
Figure 18 is presented at the bar graph of the middle enzyme concn of the pyrophosphorolysis activatory polymerization (PAP) that utilizes G46E L329A D640G S671F E678G (GLDSE) CS5DNA polysaccharase to the effect of threshold circulation (Ct) value.The y axle is represented the Ct value, and the x axle is represented enzyme concn (nM).The incidental legend of chart shows that the copy number corresponding to the template nucleic acid of each bar trace among the figure (does not have template nucleic acid copy (no temp), 1e 4Individual template nucleic acid copy (1E4/rxn), 1e 5Individual template nucleic acid copy (1E5/rxn), 1e 6Individual template nucleic acid copy (1E6/rxn)).
Figure 19 is the bar graph that shows the stdn extension speed of the various mutant of hot bacterial classification Z05DNA polysaccharase of dwelling.Speed is extended in y axle representative relatively, dwell hot bacterial classification Z05 archaeal dna polymerase (Z05) and have the suddenly change various Z05 archaeal dna polymerases of (Q=T541R, D=D580G, and S=A610F) of specified point of x axle representative.The x axle is also represented ES112 (E683R Z05 archaeal dna polymerase, the Application No. 20020012970 of " the High temperature reversetranscription using mutant DNA polymerases " by name that equals to submit to March 30 calendar year 2001 referring to Smith) and ES112-D (D580G E683R Z05 archaeal dna polymerase).The extension rate value that the mutant polysaccharase obtains comes stdn with respect to the extension rate value that the Z05 archaeal dna polymerase that is made as 1.00 obtains.
Figure 20 shows to relate to the gel photograph that the titrimetric PCR product of the relevant HIV dna profiling of PAP detects.
Figure 21 is the observed threshold circulation of the various mutant K-Ras plasmid template copy numbers (C that shows the amplification of (blocked) or nonocclusive (unblocked) primer of being used to comprise sealing T) value chart.
Figure 22 is various enzymes and the observed threshold circulation of the enzyme concn (C that shows the amplification that is used to comprise the K-Ras plasmid template T) value chart.
Figure 23 is the bar graph that shows the PAP reverse transcription reaction data of HCV RNA, and wherein the cDNA reaction product uses the special quantitative PCR analysis of HCV cDNA to measure.The y axle is represented the Ct value, the unit of enzyme number that the representative of x axle is used to react.As what point out, the enzyme that uses in these reactions is the mixture of Z05 archaeal dna polymerase (Z05) or G46E L329A Q601R D640G S671FE678G (GLQDSE) and G46E L329A Q601R D640G S671F (GLQDS) CS5DNA polysaccharase.
Figure 24 shows the PCR growth curve of the BRAF oncogene amplification that produces when carrying out two-way PAP.The accumulation fluorescence of x axle display standardization, the y axle shows the cycle number of PAP pcr amplification.
Detailed Description Of The Invention
The invention provides have in the Polymerase Structure territory one or more amino acid with respect to functional dna polymerase mutant new mutant DNA polymerases. Mutant DNA polymerase of the present invention is that the nucleotides that the form not modified with respect to polymerase has an improvement mixes the organized enzyme of speed, also has the reverse transcriptase activity followed and/or the increase of amplification ability in specific embodiment. Can under lower concentration, use mutant DNA polymerases, reach above parent enzyme or the performance equivalent with it. In specific embodiment, mutant DNA polymerase described herein has the heat endurance of improvement with respect to parent enzyme. Therefore mutant DNA polymerases is useful to relating to primer extension and the reverse transcription of polynucleotide template or the multiple application of amplification, comprises, for example the application in the medical diagnosis of recombinant DNA research and disease.
Be suitable for having the functional polyalkylene synthase domain that comprises following amino acid motif according to the not modified form of the archaeal dna polymerase of sudden change of the present invention:
(a)
Xaa-Xaa-Xaa-Xaa-Arg-Xaa-Xaa-Xaa-Lys-Leu-Xaa-Xaa-Thr-Tyr-Xaa-Asp
(also be called with the single-letter coding herein
X a1-X a2-X a3-X a4-R-X a6-X a7-X a8-K-L-X a11-X a12-T-Y-X a15-X a16(SEQ ID NO:1)); Wherein
X a1Ile (I) or Leu (L);
X a2Gln (Q) or Leu (L);
X a3Gln (Q), His (H) or Glu (E);
X a4Tyr (Y), His (H), or Phe (F);
X a6Glu (E), Gln (Q) or Lys (K);
X a7Ile (I), Leu (L) or Tyr (Y);
X a8Gln (Q), Thr (T), Met (M), Gly (G) or Leu (L);
X a11Lys (K) or Gln (Q);
X a12Ser (S) or Asn (N);
X a15Ile (I) or Val (V); And
X a16Glu (E) or Asp (D);
(b)Thr-Gly-Arg-Leu-Ser-Ser-Xaa-Xaa-Pro-Asn-Leu-Gln-Asn
(also be called with the single-letter coding herein
T-G-R-L-S-S-X b7-X b8-P-N-L-Q-N (SEQ ID NO:2)); Wherein
X b7Ser (S) or Thr (T);
X b8Asp (D), Glu (E) or Asn (N); With
(c)Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Asp-Tyr-Ser-Gln-Ile-Glu-Leu-Arg
(also be called with the single-letter coding herein
X c1-X c2-X c3-X c4-X c5-X c6-X c7-D-Y-S-Q-I-E-L-R (SEQ ID NO:3); Wherein
X c1Gly (G), Asn (N) or Asp (D);
X c2Trp (W) or His (H);
X c3Trp (W), Ala (A), Leu (L) or Val (V);
X c4Ile (I) or Leu (L);
X c5Val (V), Phe (F) or Leu (L);
X c6Ser (S), Ala (A), Val (V) or Gly (G); And
X c7Ala (A) or Leu (L).
These motifs are present in the archaeal dna polymerase that many families Aform DNAs relies on, especially from about 100 amino acid whose zones of the avtive spot of the heat-stable DNA polymerase of Thermophilic Bacteria. For example, Fig. 1 shows the amino acid sequence comparison from the zone in the archaeal dna polymerase Polymerase Structure territory of several Thermophilic Bacteria: Thermotoga maritima, thermus aquaticus, thermus thermophilus, Huang dwell hot bacterium, the thread hot bacterium that dwells, the hot bacterial classification sps17 of dwelling, the hot bacterial classification Z05 of dwelling, Naples dwell thermobacillus, Africa dwell hot chamber bacterium, Bacillus caldotenax and Thermus caldophilus. The amino acid sequence comparison that Fig. 1 shows also comprises the zone from representational chimeric heat-stable DNA polymerase Polymerase Structure territory. As shown, SEQ ID NOs.1,2 and 3 each motif all are present in each of these polymerases centres, show the conservative function in these avtive spot zones.
Accordingly, in some embodiments, the not modified form of archaeal dna polymerase is wild type or naturally occurring archaeal dna polymerase, for example, and from the polymerase of any kind above-listed Thermophilic Bacteria. In a mutation, not modified polymerase comes from the kind of Thermus. In other embodiment of the present invention, not modified polymerase comes from the thermophilic bacterial classification except Thermus. The nucleic acid of many heat-stable DNA polymerases and amino acid whose complete sequence are obtainable. The dwell sequence of hot chamber bacterium (Taf) polymerase of thermus aquaticus (Taq), thermus thermophilus (Tth), the hot bacterial classification Z05 of dwelling, the hot bacterial classification sps17 of dwelling, Thermotoga maritima (Tma) and Africa is announced in pct international patent publication number WO 92/06200. Announced (Nucleic Acids Research 20:5839,1992) by Akhmetzjanov and Vakhitov from the dwell sequence of archaeal dna polymerase of hot bacterium of Huang. Sequence from the heat-stable DNA polymerase of Thermus caldophilus is found at EMBL/GenBank accession number U62584. The sequence information that can use method that U.S. Patent number 4,889,818 for example provides and table 1 to provide from the sequence of the heat-stable DNA polymerase of the thread hot bacterium that dwells regains from ATCC preserving number 42380. The dwell sequence of thermobacillus archaeal dna polymerase of Naples comes from GeneSeq patent database accession number R98144 and PCT WO 97/09451. From the sequence of the heat-stable DNA polymerase of Bacillus caldotenax by describe (J BioChem (Tokyo) 113 (3): 401-410,1993 such as Uemori etc.; Also referring to Swiss-Prot database login Q04957 and GenBank accession number D12982 and BAA02361). Can also be described in such as the example of the not modified form of the archaeal dna polymerase of describe modifying herein, for example, authorize the U.S. Patent number 6,228,628 of Gelfand etc. " Mutant chimeric DNA polymerase " by name May 8 calendar year 2001; Authorized on February 12nd, 2002 Gelfand etc. " Thermostable DNA polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes " by name 6,346,379; Authorized on April 18th, 2006 Ankenbauer etc. " Thermostable enzyme promoting the fidelity of thermostable DNA polymerases-for improvement of nucleic acid synthesis and amplification in vitro " by name 7,030,220; Authorized on April 19th, 2005 Sobek etc. " Mutant B-type DNA polymerases exhibiting improved performance in PCR " by name 6,881,559; Authorized on September 21st, 2004 Markau etc. " Modified DNA-polymerase from carboxydothermus hydrogenoformans and its use for coupled reverse transcription and polymerase chain reaction " by name 6,794,177; Authorized on October 22nd, 2002 Ankenbauer etc. " Thermostable DNA polymerase from carboxydothermus hydrogenoformans " by name 6,468,775; And Schoenbrunner equals the Application No. 20040005599 of " Thermostableor thermoactive DNA polymerase molecules with attenuated 3 '-5 ' exonuclease activity " by name of submission on March 26th, 2003; 20020012970 of " the High temperature reverse transcription using mutant DNA polymerases " by name that Smith equals to submit to March 30 calendar year 2001; 20060078928 of " the Thermostable enzyme promoting the fidelity of thermostable DNA polymerases-for improvement of nucleic acid synthesis and amplification in vitro " by name that Ankenbauer equals to submit on September 29th, 2005; 20040115639 of " the Reversibly modified thermostable enzymes for DNA synthesis and amplification in vitro " by name that Sobek equals to submit on December 11st, 2002.
The functional DNA polymerase of modified (for example by 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, insertion or disappearance) is suitable for sudden change described herein equally as long as keep SEQ ID NOs.1,2 and 3 amino acid motif in advance. Therefore, suitable not modified archaeal dna polymerase also comprises the functional variant of wild type or naturally occurring polymerase. Above-mentioned variant usually and wild type or naturally occurring polymerase have basically sequence homogeneity or similitude, typically at least 80% sequence homogeneity, more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homogeneity. In specific embodiment, not modified archaeal dna polymerase have reverse transcriptase (RT) active and/or mix ribonucleotide or other 2 '-ability of the nucleotides modified.
Suitable polymerase also comprises, for example, comprises the specific chimeric dna polymerase from the polypeptide zone of two or more enzymes. The example of above-mentioned chimeric dna polymerase is described in for example U.S. Patent number 6,228,628. Specially suitable is chimeric CS family archaeal dna polymerase, it comprise CS5 (SEQ ID NO:18) and CS6 (SEQ ID NO:19) polymerase and with SEQ ID NO:18 or SEQ ID NO:19 have basically sequence homogeneity or the variant (typically at least 80% sequence homogeneity, more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homogeneity) of similitude. CS5 and CS6DNA polymerase are to derive from dwell hot bacterial classification Z05 and the Thermotoga maritima ((chimaeric enzyme of (Tma) archaeal dna polymerase. They comprise 5 of the hot bacterium enzyme N-end of dwelling '-3 '-5 ' exonuclease and the Polymerase Structure territory of the C-end of nuclease domain and Tma enzyme. These enzymes have effective reverse transcriptase activity, can extend the primer that comprises nucleotide analog, and can mix the dNTPs of α-D2EHDTPA dNTPs, dUTP, dITP and fluorescein and cyanine dye family mark. CS5 and CS6 polymerase or effective Mg2+The PCR enzyme of activation. The nucleotide sequence of coding CS5 and CS6 polymerase provides in Fig. 2 B and 3B respectively. The chimeric polymerase of CS5 and CS6 is further described in, for example in the U.S. Patent Application Publication No. 2004/0005599.
In some embodiments, the not modified form of archaeal dna polymerase is the polymerase of having modified in advance, and the typical case is through restructuring, to give certain selective advantage. Above-mentioned modification comprises, for example, and 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor G46E, L329A and/or E678G in CS5 archaeal dna polymerase, the CS6 archaeal dna polymerase, or the corresponding sudden change in other polymerase. Accordingly, in specific mutation, the not modified form of DNA polymerase is following a kind of (amino acid sequence that all has separately SEQ ID NO:18 or SEQ ID NO:19 except the replacement of appointment): G46E; G46E L329A; G46E E678G; Or G46E L329A E678G. E678G replaces, and for example, allows the mixing of nucleotides of ribonucleotide and other 2 ' modify, but this sudden change appears also to cause extending the weakening of the ability of the template that has caused. In specific embodiment, the present invention who improves saltant polymerase Drawing rate suddenlys change and has improved this special character of E678G sudden change.
With respect to not modified polymerase, mutant DNA polymerases of the present invention comprises one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at avtive spot. In some embodiments, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is at least one at following amino acid sites:
The site X of the motif of SEQ ID NO:1 regulationa8；
The site X of the motif of SEQ ID NO:2 regulationb8；
The site X of the motif of SEQ ID NO:3 regulationc4 With
The site X of the motif of SEQ ID NO:3 regulationc6。
One or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in these sites provides the nucleotides of improvement to mix activity, obtains having with respect to not modified polymerase the mutant DNA polymerases of (faster) nucleic acid Drawing rate of improvement. In addition, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in one or more these sites provides 3 '-5 ' exonuclease (correction) activity that increases with respect to not modified polymerase. Be not limited to any specific theory, the inventor believes that the nucleic acid Drawing rate of the improvement of saltant polymerase of the present invention is the result who more combines closely with template, namely less frequently dissociates from template, causes the enzyme of higher " processivity ". These character are allowed in primer extension reaction for example, with respect to the reaction that relates to not modified archaeal dna polymerase, use the saltant polymerase of lower concentration. Therefore, can imagine ground, under sufficiently high enzyme concentration, the Drawing rate of not modified polymerase (namely lacking the specific sudden change of theme of the present invention) can be near the Drawing rate of mutant enzyme. The performance of saltant polymerase appears also greatly to be better than not modified form under high ionic strength. However, under low ionic strength, enough the performance of the not modified polymerase of high enzyme concentration may be near the performance of saltant polymerase.
Because the not modified form of archaeal dna polymerase is single, for each saltant polymerase, corresponding to Xa8、X b8、X c4And Xc6In each amino acid sites typical case be different. Amino acid and nucleotide sequence comparison program can easily obtain (referring to, for example mentioned above those), with in the situation that provides the specific motif of identifying herein, with the evaluation of helping the definite amino acid (with corresponding codon) according to modification of the present invention. Representational chimeric heat-stable DNA polymerase and from the heat-stable DNA polymerase of exemplary Thermophilic Bacteria species corresponding to Xa8、X b8、X c4And Xc6In each site be shown in table 1.
Table 1. in exemplary heat-stabilised poly synthase corresponding to motif site Xa8、X b8、X c4And Xc6Amino acid sites.
In some embodiments, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is single 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. The saltant polymerase can, for example, be included in respectively site Xa8、X b8、X c4Or Xc6Any one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. Selectively, the saltant polymerase is included in various combinations any of 2,3 or all 4 replacements in these sites. For example, in one embodiment, mutant DNA polymerases of the present invention comprises site Xb8And Xc6Each 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. Typically, at site Xa8、X b8、X c4Or Xc6Amino acid with the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the motif of not stipulating separately corresponding to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Therefore, typically, at site Xa8Amino acid if to be substituted be not Q, T, M, G or L so; At site Xb8Amino acid if to be substituted be not D, E or N so; At site Xc4Amino acid if to be substituted be not I or L so; And/or at site Xc6Amino acid if to be substituted be not S, A, V or G so. In specific embodiment, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is included in site Xa8Arginine (R), at site Xb8Glycine (G), at site Xc4Phenylalanine (F), and/or at site Xc6Phenylalanine (F). Other suitable 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in the site of one or more evaluation can use, example as is known direct mutagenesis method and further describe herein or in addition in the test known to those skilled in the art the mensuration of primer extension property determine.
As previously discussed, in some embodiments, mutant DNA polymerases of the present invention stems from CS5 archaeal dna polymerase (SEQ ID NO:18), CS6 archaeal dna polymerase (SEQ ID NO:19), or the variant of those polymerases (for example, G46E; G46E L329A; G46E E678G; G46E L329A E678G etc.). As top mentioned, in CS5 archaeal dna polymerase or CS6 archaeal dna polymerase, site Xa8Corresponding at 601 glutamine (Q); Site Xb8Corresponding at 640 aspartic acid (D); Site Xc4Corresponding at 669 isoleucine (I); And site Xc6Corresponding at 671 serine (S). Therefore in specific mutation of the present invention, with respect to CS5 archaeal dna polymerase or CS6 archaeal dna polymerase, the saltant polymerase comprises at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at S671, D640, Q601 and/or I669. Exemplary CS5 archaeal dna polymerase and CS6 dna polymerase mutant body comprise the mutant that contains 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor S671F, D640G, Q601R and/or I669F. In some embodiments, saltant CS5 polymerase or saltant CS6 polymerase comprise, for example, and the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (for example D640G and S671F) at D640 and S671 two places. The CS5 archaeal dna polymerase that other is exemplary and CS6 dna polymerase mutant body comprise following (amino acid sequence that has separately SEQ ID NO:18 or SEQ ID NO:19 except the replacement of appointment):
G46E S671F；
G46E D640G；
G46E Q601R；
G46E I669F；
G46E D640G S671F；
G46E L329A S671F；
G46E L329A D640G；
G46E L329A Q601R；
G46E L329A I669F；
G46E L329A D640G S671F；
G46E S671F E678G；
G46E D640G E678G；
G46E Q601R E678G；
G46E I669F E678G；
G46E D640G S671F E678G；
G46E Q601R D640G S671F E678G；
G46E Q601R D640G S671F I669F E678G；
G46E L329A S671F E678G；
G46E L329A D640G E678G；
G46E L329A Q601R E678G；
G46E L329A I669F E678G；
G46E L329A D640G S671F E678G; With
G46E L329A Q601R D640G S671F E678G.
Except that the sudden change of the motif of SEQ ID NOs 1,2 described herein and/or 3, mutant DNA polymerases of the present invention can also comprise the modification of other non-replacement.Above-mentioned modification can comprise that for example, covalent modification known in the art is with the additional advantage in the application that comprises primer extension.For example, in specific embodiment, mutant DNA polymerases further comprises hot reversible covalent modification.In these embodiments, the modifier group is covalently attached to protein, cause the loss all or almost all of enzymic activity.The modifier group is through selecting, so that hatch this modification of reverse through high temperature.Comprise that the reversibly modified archaeal dna polymerase of above-mentioned heat is particularly suitable for the application of hot start, for example, the round pcr of various hot starts.Be suitable for being described in according to the hot reversibly modified thing reagent that mutant DNA polymerases of the present invention is used, for example, the U.S. Patent number 5,773,258 of Birch etc.Exemplary modification comprises, for example, and the reversible sealing of lysine residue of the chemically modified of the epsilon-amino by lysine residue (referring to above Birch etc.).In specific mutation, hot reversible covalent modification comprises that the dicarboxylic anhydride of descriptions such as Birch as mentioned is covalently attached to the epsilon-amino of lysine residue.
For example, the specially suitable mutant polysaccharase that comprises hot reversible covalent modification is by thermophilic enzyme with have mixture as the dicarboxylic anhydride of the general formula of following formula I regulation at alkaline pH be lower than the reaction of carrying out under about 25 ℃ temperature and produce:
Figure A20078003877200341
R wherein 1And R 2Be hydrogen or organic group, they can connect together; Or have a following formula II:
R wherein 1And R 2Be organic group, they can connect together, and hydrogen is cis, and are described as Birch etc. basically.In comprising the specific embodiment of hot reversible covalent modification, be G64E CS5 archaeal dna polymerase without the polysaccharase of modified forms.
Mutant DNA polymerases of the present invention can be by the corresponding not modified polysaccharase of sudden change coding (for example, the variation that wild-type polysaccharase or mutant polysaccharase of the present invention are derived from) dna sequence dna makes up, and for example is commonly referred to the technology of site-directed mutagenesis by use.Coding can suddenly change by various polymerase chain reactions (PCR) technology that those of ordinary skills know without the nucleic acid molecule of the polysaccharase of modified forms.(referring to, for example, PCRStrategies (M.A.Innis, D.H.Gelfand, and J.J.Sninsky eds., 1995, Academic Press, San Diego, CA) the 14th chapter; PCR ProtoCols:A Guide toMethods and Applications (M.A.Innis, D.H.Gelfand, J.J.Sninsky, and T.J.White eds., Academic Press, NY, 1990).
As limiting examples, the two primer systems that use from the conversion site site-directed mutagenesis test kit of Clontech can be used for rite-directed mutagenesis is introduced the polynucleotide of the not modified form of polysaccharase of encoding.After the target plasmid sex change in the system, two primers are annealed to plasmid simultaneously; A rite-directed mutagenesis that comprises needs in the primer, another is included in another site mutation that causes in this plasmid that restriction site is eliminated.Then carry out the synthetic of second chain, closely connect this two sudden changes, then the plasmid that produces is transformed into intestinal bacteria mutS bacterial strain.Plasmid DNA is separated from the bacterium that transforms, and the restriction enzyme restriction enzyme digestion (thereby plasmid that linearizing does not suddenly change) with relevant is transformed into intestinal bacteria then again.This system allows directly to produce sudden change on expression plasmid, and needn't subclone or produce the phasmid of strand.The linearizing of the tight connection of two sudden changes and not mutant plasmid subsequently causes high mutation efficiency and allows minimum screening.After initial limit site primer synthetic, only need use a kind of new primer type to this method of each mutational site.Not the mutant of preparing each site respectively, can synthesize one group of " degeneracy of design " Oligonucleolide primers, be used for introducing in given site simultaneously the sudden change of all needs.Transformant can check order by the plasmid DNA to the mutagenic treatment zone and screen to identify and the classification mutant clone.Every electrophoretic analysis is cut and used to being limited property of mutant DNA enzyme, for example, detect enhancing gel (Mallinckrodt Baker in sudden change, Inc., Phillipsburg carries out on NJ) to confirm not have on this sequence other change to have (moving comparison by being with the contrast of mutagenic treatment not).Perhaps can be to the order-checking of whole DNA zone to confirm outside target region, not having other catastrophic event to take place.
Cross the mutant duplex of verifying on the expression vector at pET (perhaps other) and can be used for transformed into escherichia coli, coli strain BL21 (DE3) pLysS for example reaches the high level production of mutant protein, and comes purifying with standard schedule.The method of FAB-MS mapping for example, can be used for the fidelity of reproduction that the quick check mutant is expressed.This technology provides and spreads all over whole proteinic segmental order-checking and degree of confidence necessary in the order-checking task is provided.In the mapping of the type experiment, protein with protease digestion (selection will be depended on the specific region that will modify because this fragment be major concern and remaining collection of illustrative plates should with the proteinic collection of illustrative plates of mutagenic treatment is not consistent).Cutting segment group is passed through, and for example microbore HPLC (specific region that will modify is depended in anti-phase or ion-exchange equally) fractional separation is to be provided at some peptides of each several part, and the molecular weight of these peptides is measured such as FAB-MS with standard method.The molecular weight ratio of the peptide of the expectation that obtains of the quality that each fragment is measured and forecasting sequence digestion and is determined the exactness that checks order fast then.Fix a point because carry out the mutagenesis of protein modification, if the MS data conform to prediction, the order-checking of the peptide of change is dispensable.The residue that changes of checking if necessary, CAD-series connection MS/MS can be used for the order-checking of the peptide of the mixture discussed, and perhaps the target peptide can be purified and subdues Edman degraded or carboxypeptidase y digestion according to modifying the position.
Mutant DNA polymerases with an above aminoacid replacement can produce with multiple mode.Be positioned on the polypeptide chain under the situation (as the amino acid Xc4 and the Xc6 of the motif of SEQ ID NO:3 regulation) thick and fast at amino acid, they can use an oligonucleotide of all aminoacid replacement that need of coding to come simultaneous mutation.If yet amino acid mutually positioning (for example being separated with upper amino acid) spaced apart by 10, the single oligonucleotide that produces all variations that need of coding is more difficult.As an alternative, can be with in two kinds of optional methods a kind of.In first method, each amino acid that will replace is produced isolating oligonucleotide.Then oligonucleotide is annealed on the single-stranded template DNA simultaneously, and from template synthetic DNA second chain all aminoacid replacement that need of will encoding.An optional method relates to two or more round-robin mutagenesis to produce the mutant of needs.First circulation is as described in the single mutant: the DNA of the not modified polysaccharase of encode is used as template, and the oligonucleotide of the aminoacid replacement of first needs of coding is annealed on this template, produces the heteroduplex DNA molecule then.Second round-robin mutagenesis utilizes the mutant DNA of first round-robin mutagenesis generation as template.Therefore, this template has comprised one or more sudden change.The oligonucleotide of aminoacid replacement of other needs of will encoding then is annealed to this template, then the DNA chain that produces now coding from the sudden change of first and second round-robin mutagenesis.The DNA that produces can be used as template in the 3rd round-robin mutagenesis, or the like.Perhaps can utilize the multidigit point mutagenesis (Anal.BioChem.324:285-291.2004) of Seyfang and Jin.
The recombinant nucleic acid of coding any mutant DNA polymerases of the present invention correspondingly, also is provided.Use the nucleic acid of encoding mutant type archaeal dna polymerase of the present invention can produce various carriers.Comprise the replicon that derives from the species compatible and any carrier of control sequence and can be used to implement the present invention with host cell.Usually, expression vector comprises with the nucleic acid of encoding mutant type archaeal dna polymerase and can handle transcribing and the translational control nucleic acid region of being connected.It is the required dna sequence dna of expression that can handle the encoding sequence of connection that term " control sequence " refers in specific host organisms.Be applicable to procaryotic control sequence, for example, comprise promotor, randomly operator gene sequence, and ribosome bind site.In addition, carrier can comprise forward and reverse controlling element (PositiveRetroregulatory Element, PRE) transformation period (referring to the U.S. Patent number 4,666,848 of Gelfand etc.) of the mRNA that transcribes with raising.Transcribe the host cell that is generally suitable for being used for expressing polysaccharase with the translational control nucleic acid region.Many suitable expression vector and the proper regulation sequence that is used for various host cells known in the art.Substantially, transcribing and translate the adjusting sequence can comprise, for example, and promoter sequence, ribosome bind site, transcription initiation and terminator sequence, translation initiation and terminator sequence and enhanser or activation sequence.In typical embodiment, regulate sequence and comprise promotor and transcription initiation and terminator sequence.Carrier typically also comprises and contains some polylinker zones that are used for the restriction site that foreign DNA inserts.In specific embodiment, " merging sign " is used to help purifying and the removal of label/flag sequence subsequently (if necessary), for example " histidine-tagged (His-Tag) ".Yet these are normally unnecessary when purifying hot activation and/or heat stable protein from have a liking for Wensu master (for example intestinal bacteria), and it can use " hot step (heat-step) ".Comprise the coding replication sequence, regulate sequence, the structure of the suitable carriers of the DNA of Phenotypic Selection gene and the mutant polysaccharase be concerned about uses the preparation of standard recombinant dna program.Isolating plasmid, virus vector and dna fragmentation with specific order cutting, cutting and connect together carrier produce to need, it is known in the art (referring to, for example, Sambrook etc., Molecular Cloning:A Laboratory Manual (Cold SpringHarbor Laboratory Press, New York, NY, 2nd ed.1989)).
In specific embodiment, expression vector comprises that the selected marker is to allow the selection of transformed host cells.It is known in the art selecting gene, and it becomes with the host cell that uses.Suitable selection gene can comprise, the gene of for example encode penbritin and/or tetracyclin resistance, and it makes with these carrier cell transformed and can grow under the situation that these microbiotic exist.
In one aspect of the invention, the nucleic acid of encoding mutant type archaeal dna polymerase is to be introduced into cell separately or with carrier-bound form." introducing " or phraseological Equivalent represent that the mode that nucleic acid is integrated, increased and/or express with a kind of nucleic acid that is suitable for subsequently enters cell herein.The method of introducing is mainly arranged by target cell type.Exemplary method comprises CaPO 4Precipitation, liposome merges,
Figure A20078003877200371
Electroporation, virus infection etc.
Prokaryotic organism are used as the host cell of the initial clone's step of the present invention usually.They are used as the production of the single stranded DNA of site-directed mutagenesis template for the rapid production of a large amount of DNA, and the dna sequencing of mutant that screens many mutant and generation simultaneously is particularly useful.Suitable prokaryotic host cell comprises e. coli k12 strain 94 (ATCC No.31,446), coli strain W3110 (ATCC No.27,325), e. coli k12 strain DG116 (ATCC No.53,606), intestinal bacteria X1776 (ATCC No.31,537) and intestinal bacteria B; Yet colibacillary many other bacterial strains such as HB101, JM101, NM522, NM538, NM539, and many other kinds and the prokaryotic organism that belong to comprise bacillus such as Bacillus subtillis (Bacillussubtilis), all can both be used as the host such as other enterobacteriaceae of salmonella typhimurium (Salmonella typhimurium) or serratia marcescens (Serratia marcesans) and the bacterial classification of various Rhodopseudomonas (Pseudomonas species).The calcium chloride methods that the host cell typical case that prokaryotic host cell or other have a rigidity cell walls uses Sambrook as mentioned etc. to describe at 1.82 joints transform.Perhaps can adopt electroporation to transform these cells.The prokaryotic organism transformation technology is set forth in, for example, and Dower, in Genetic Engineering, Principles andMethods 12:275-296 (Plenum Publishing Corp., 1990); Hanahan etc., Meth.Enzymol., 204:63,1991.The typical case is used for the plasmid that intestinal bacteria transform and comprises pBR322, pUCI8, pUCI9, pUCI18, pUC119 and Bluescript M13, they all be described in the 1.12-1.20 part of above Sambrook etc.Yet many other suitable carriers also are available.
Mutant DNA polymerases typical case of the present invention be fit to induce or condition that mutagenesis type archaeal dna polymerase is expressed under produce by cultivating with the expression of nucleic acids carrier transformed host cells that comprises encoding mutant type archaeal dna polymerase.In the method that is suitable under the condition of protein expression cultivating transformed host cells is (referring to, for example, Sambrook above etc.) known in the art.Be used for comprising that from the proper host cell of the plasmid vector production mutant polysaccharase that comprises λ pL promotor coli strain DG116 (ATCC No.53606) is (referring to U.S. Patent number 5,079,352 and Lawyer, F.C. etc., PCR Methods and Applications 2:275-87,1993).After expressing, can gather in the crops and the segregation mutant polysaccharase.The method of purifying heat-stable DNA polymerase is described in, for example, and Lawyer above etc.
Behind the purifying, mutant DNA polymerases extends the ability of the template caused can be tested being used for measuring any various known tests that Nucleotide mixes.For example, under the situation that the template molecule (for example M13 DNA etc.) that has caused, suitable damping fluid, a whole set of dNTPs (for example dATP, dCTP, dGTP and dTTP) and metal ion are arranged, archaeal dna polymerase will extend primer, and single stranded DNA (ssDNA) is changed into double-stranded DNA (dsDNA).Transformation can be passed through, and the dyestuff in conjunction with dsDNA that for example adds such as SYBR Green I detects with quantitative.Use dynamic thermal cycler (referring to, Watson, etc. Anal.BioChem.329:58-67,2004, also can obtain from for example Applied Biosystems, Stratagene and BioRad), can obtain the digitized video (for example) of Sptting plate, thereby allow the improvement of subsequent reactions with the 10-30 interval of second.The fluorescence numerical value that detects can easily change extension speed into.Use above-mentioned routine test, can measure the extension speed of the mutant form not modified with respect to polysaccharase.
Mutant DNA polymerases of the present invention can be used for any purpose that wherein above-mentioned enzymic activity is necessary or needs.Correspondingly, in another aspect of the present invention, provide the primer extension method that uses the mutant polysaccharase.The condition that is suitable for primer extension is known in the art.(referring to, for example, Sambrook above etc.Also referring to Ausubel etc., Short Protocols inMolecular Biology (4th ed., John Wiley﹠amp; Sons 1999)).Usually, primer annealing, promptly hybridize to target nucleic acid to form primer-template composite.In suitable environment with primer-template composite with the contact of mutant DNA polymerases and free nucleotide, allow one or more Nucleotide append to 3 of primer '-end, thereby produce the primer that extends with the target nucleic acid complementary.Primer can comprise, for example, and one or more nucleotide analog.In addition, free nucleotide can be conventional Nucleotide, unconventional Nucleotide (for example Nucleotide of ribonucleotide or mark) or its mixture.In some mutation, primer extension reaction comprises the amplification of target nucleic acid.Being suitable for using archaeal dna polymerase and primer also is (for example pcr amplification method) known in the art to the condition of carrying out nucleic acid amplification.(referring to, for example, Sambrook above etc.; Ausubel above etc.; PCR Applications:ProtoCols for Functional Genomics (eds. such as Innis, Academic Press 1999)).In other non-exclusive embodiment, primer extension reaction comprises the reverse transcription (for example RT-PCR) of RNA template.Provide the use of this mutant polysaccharase of the extension speed of improvement to allow, for example, with short relatively incubation time, the enzyme concn of reduction and/or the product yield of increase are implemented the ability of above-mentioned primer extension reaction.
In other embodiments, the mutant polysaccharase is used for the primer extension under the background of dna sequencing, dna marker or primer extension product mark.For example, use the dna sequencing of Sanger dideoxyribonucleoside acid system (Sanger etc., ProC.Natl.Acad.Sci.USA 74:5463,1977) to be improved, because polysaccharase can mix unconventional chain termination nucleotide by the present invention.The development of the method for the Sanger on basis etc. provides new carrier (Yanisch-Perron etc., Gene33:103-119,1985) and base analogue (Mills etc., ProC.Natl.Acad.Sci.USA76:2232-2235,1979; With Barr etc., Biotechniques 4:428-432,1986).In general, dna sequencing need produce the segmental distribution of separated portions by size subsequently at the primer extension of the dependence of the template under the situation that the chain termination base analogue is arranged.The dideoxy sequencing program on basis comprises that the Oligonucleolide primers that (i) will randomly be labeled is annealed to template; (ii) in 4 isolating reactions, extend primer, the mixture that each reaction comprises unlabelled dNTPs and limits to a number or amount such as the chain terminator that randomly is labeled of ddNTP with archaeal dna polymerase; (iii) on high-resolution denaturing polyacrylamide/urea gel, separate 4 group reaction products.Reaction product can detect on gel by radioautography or by fluoroscopic examination, depends on the mark of use, can check that then image is to infer nucleotide sequence.These methods are utilized the archaeal dna polymerase such as the T7 archaeal dna polymerase of the Klenow fragment of intestinal bacteria Pol I or modification.
The operability of heat-stabilised poly synthase (for example Taq archaeal dna polymerase), the sequence measurement with heat-stable DNA polymerase that has caused improvement is (referring to Innis etc., ProC.Natl.Acad.Sci.USA 85:9436,1988) and be called its process variant (Murray of " cycle sequencing ", Nuc AcidsRes.17:8889,1989).Mutant heat-stabilised poly synthase correspondingly of the present invention can be used to described method.As substituting of the dideoxy method on basis order-checking, cycle sequencing is in the amplification linearity that the target sequence that is complementary to template sequence under the situation of chain terminator is arranged, asymmetric.Single circulation produces the family of the extension products of the possible length of institute.After the dna profiling sex change, multicycle primer annealing and primer extension take place in the presence of such as the terminator of ddNTPs continue the extension product.Cycle sequencing only need be than the chain termination sequencing template DNA still less of routine.Heat-stable DNA polymerase has some advantages in cycle sequencing; The harsh annealing temperature of the specific cross needs of their tolerance primer and nucleic acid target and tolerate that multicycle to occur in each round-robin high-temperature denatured, for example 90-95 ℃.For this reason, Archaeal dna polymerase and derivative thereof and descendant, for example AmpliTaq CS archaeal dna polymerase and AmpliTaq FS archaeal dna polymerase have been included in (the Norwalk such as Perkin-Elmer, CT) and Applied Biosystems (FosterCity is in the business-like Taq cycle sequencing test kit of company CA).
The mutation of chain termination sequencing method comprises dyestuff-primer order-checking and dyestuff-terminator order-checking.In dyestuff-primer order-checking, the ddNTP terminator is unlabelled, and the primer of mark is used for detecting extension products (Smith etc., Nature 32:674-679,1986).In dyestuff-terminator dna sequencing, archaeal dna polymerase is used for mixing dNTPs and fluorescently-labeled ddNTPs (Lee etc., Nuc.Acids.Res.20:2471,1992) on the end of primed DNA.This process provides the advantage of primer that needn't the synthetic dyestuff marks.In addition, to be reflected at that whole 4 reactions can carry out in same pipe on this point be more easily to dyestuff-terminator.
Dyestuff-primer and dyestuff-terminator method both can use Applied Biosystems (FosterCity, the CA) automatic sequencer of Sheng Chaning (U.S. Patent number 5,171,534) operation automatically.When using this instrument, the sequencing reaction mixture of finishing is at denaturing polyacrylamide gel or be contained in fractional separation on the kapillary on this instrument.Fluorescence-causing substance is with the laser detection of instrument bottom, because they pass through gel according to big or small electrophoresis.
Two kinds of fluorescence dyes are commonly used to the terminator that mark is used for dyestuff-terminator order-checking--electronegative and zwitterionic fluorescence dye.Electronegative fluorescence dye comprises the fluorescence dye of fluorescein and BODIPY family.The glimmering dyestuff of fluorine boron (BODIPY dyes, 4,4-two fluoro-4-boron-3a, 4a-diaza-s-indacene) are described in international patent publications WO 97/00967.The zwitter-ion fluorescence dye comprise those of rhodamine family.Commercially available cycle sequencing test kit uses the terminator of rhodamine derivative mark.Yet the terminator of rhodamine mark is quite expensive and product must separate from uncorporated dyestuff-ddNTPs before last sample is to the gel, because they move jointly with the product that checks order.As if rhodamine family terminator makes the hairpin structure in the zone of being rich in GC stable, causes the unusual migration of product.This need use dITP, and it makes secondary structure loose and influence the efficient that terminator mixes.
On the contrary, fluorescein-labeled terminator has been removed separating step before the sample on gel, because they have bigger net negative charge and move sooner than the order-checking product.In addition, fluorescein-labeled order-checking product has better electrophoretic migration than the order-checking product of rhodamine mark.Although wild-type Taq archaeal dna polymerase can not mix the terminator of fluorescein family dye marker effectively, can finish effectively by the enzyme that utilizes the modification of describing as U.S. Patent Application Publication No. 2002/0142333 now.Correspondingly, the modification of describing as US2002/0142333 can be used to background of the present invention and has the heat-stabilised poly synthase that the fluorescein family dyestuff of the primer extension speed of improvement mixes with generation.For example, in specific embodiment, according to not modified archaeal dna polymerase of the present invention the heat-stabilised poly synthase of the modification described as US2002/0142333 and motifs with SEQ IDNOs.1,2 and 3 regulations.
Can use other exemplary nucleic acid sequencing mode of mutant DNA polymerases of the present invention to comprise therein to relate to comprise 2 of ribonucleotide '-PO 4Those of the terminator compound of analogue (referring to, the international application no WO2005/005667 that international application no WO2005/026184 that for example U. S. application publication number 2005/0037991 and 2005/0037398, and Bodepudi equals to submit on June 29th, 2004 is by name " SYNTHESIS AND COMPOSITIONS OFNUCLEIC ACIDS COMPRISING 2 '-TERMINATOR NUCLEOSIDES " and Gelfand equal to submit on June 29th, 2004 is by name " 2 '-TERMINATOR RELATED PYROPHOSPHOROLYSIS ACTIVATEDPOLYMERIZATION ").Mutant DNA polymerases described herein is common, and for example, the quantity or the concentration of the enzyme by the performance that reduces time that the round-robin extension needs and/or be used to by reduction to reach satisfied are improved these sequencing.
In another aspect of the present invention, be provided for the test kit of primer extension method described herein.Typically, test kit by partition so that use and comprise at least one container according to mutant DNA polymerases of the present invention is provided.Can also comprise the other container that one or more provides other reagent.Above-mentioned other container can comprise any reagent or other element known to the those of skill in the art of being that is used for according to the primer extension program of above-described method, comprise and being used for, for example, nucleic acid amplification program (for example PCR, RT-PCR), the reagent of dna sequencing program or dna marker program.For example, in specific embodiment, test kit further comprise be provided under the primer extension condition can with 5 of predetermined polynucleotide template hybridization ' adopted primer is arranged, or comprise 5 ' have the primer of adopted primer and corresponding 3 ' antisense primer right.In other non-exclusive mutation, test kit comprises the container that one or more provides free nucleotide (routine and/or unconventional).In specific embodiment, test kit comprises α-thiophosphoric acid (α-phophorothioate) dNTPs, dUTP, dITP, and/or the dNTPs of mark, for example fluorescein or the dNTPs of cyanine dye family.In other non-exclusive embodiment, test kit comprises the container that one or more provides the buffer reagent that is suitable for primer extension reaction.
Embodiment
Should understand, embodiment described herein and embodiment just are used for the illustrative purpose and do not mean that the scope of invention of limit request protection.Should be understood that equally, based on the various modifications of embodiment described herein and embodiment or change to be inspired and give those skilled in the art, and be included in the scope of the application's spirit and scope and additional claim.
Example I: identification and characterized with mutant DNA polymerases of improved enzymatic activity
The sudden change that provides as in the CS family polysaccharase of the downward ability of stretching primed DNA of the situation in that free nucleotide is arranged of improvement has been provided.Briefly, step in this screening procedure comprises that the library generates, the expression of mutant enzyme and partial purification, the screening of the enzyme of the character that needs, dna sequencing, the CHARACTERISTICS IDENTIFICATION of clone purification and candidate's mutant of further selecting, and from generation, purifying and the CHARACTERISTICS IDENTIFICATION of the sudden change combination of the mutant of selecting.In these steps each all is further described in hereinafter.
Sudden change by the identification of this method comprises separately or with S671F, D640G, Q601R and the I669F of various combination.These sudden changes are positioned at some CS family polysaccharase, comprise G46E CS5, G46E L329A CS5, G46E E678G CS5 and G46E L329A E678G CS5.In these mutant polysaccharases some are listed in table 2.The mutant polysaccharase that has obtained that other is exemplary comprises CS6 G46E Q601R D640G S671F E678G archaeal dna polymerase and some hot bacterial classification Z05 dna polymerase mutant body of dwelling.The mutant polysaccharase that produces is identified by the performance of analyzing them in a series of dynamic thermal cyclings (KTC) experiment.
The CS5 mutant DNA polymerases that table 2. is exemplary
G46E D640G G46E S671F E678G
G46E S671F G46E D640G S671F E678G
G46E Q601R D640G G46E Q601R D640G S671F E678G
G46E D640G S671F G46E L329A Q601R E678G
G46E Q601R D640G S671F G46E L329A D640G E678G
G46E L329A D640G G46E L329A S671F E678G
G46E L329A Q601R D640G S671F G46E L329A Q601R S671F E678G
G46E L329A S671F G46E L329A D640G S671F E678G
G46E L329A Q601R D640G G46E L329A Q601R D640G S671F E678G
G46E L329A D640G S671F G46E L329A D640G I669F S671F E678G
L329A D640G L329A Q601R E678G
L329A D640G S671F L329A S671F E678G
L329A Q601R D640G S671F S671F
L329A S671F D640G S671F
D640G Q601R D640G S671F
Cause ability through sudden change S671F, D640G, Q601R and the I669F of identification as the template that caused of extension of improvement.In the specific background of E678G sudden change, its allow ribonucleotide and other 2 '-the mixing of the Nucleotide modified, but it also causes extending the weakening of the ability of the template that has caused, the character of the primer extension ability of S671F, D640G, Q601R and this weakening of I669F sudden change the improvement.The sudden change of identification, especially independent S671F and S671F add D640G, also show the reverse transcription efficient of improvement when being arranged in G46E CS5 and G46E L329A CS5 archaeal dna polymerase.Other character of mutant DNA polymerases of the present invention is further described in down.
Clone library generates: make the nucleic acid in the Polymerase Structure territory of coding CS5 E678G archaeal dna polymerase stand fallibility (mutagenesis) PCR between the Bgl of the plasmid that comprises this nucleotide sequence II and Hind III restriction site.Use is from the Mg of the scope of 1.8-3.5mM + 2Concentration is carried out PCR, so that produce the library with respective range mutation rate.Buffer conditions is: 50mM N-two [hydroxyethyl] glycine pH 8.2,115mM KOAc, 8%w/v glycerine, every kind of dNTPs of 0.2mM and 0.2X SYBR Green I.Use GeneAmp AccuRT heat start PCR enzyme with 0.15U/ μ l.With per 50 μ l reaction volume 5x10 5The linearizing CS5E678G plasmid DNA of copy is initial, carries out 30 round-robin amplifications, uses 60 ℃ annealing temperature 15 seconds, 72 ℃ elongating temperature 45 seconds, and 95 ℃ denaturation temperature 15 seconds.
The amplicon that produces through the Qiaquick column spinner (Qiagen, Inc.Valencia, CA, USA) purifying, with Bgl II and Hind III cutting, purifying again then.The G46E L329ACS5 that vector plasmid, the Polymerase Structure territory between BglII and HindIII site carry big section disappearance modifies, by cutting with two kinds of identical restriction enzymes and preparing with calf intestinal phosphatase enzyme (CIP) processing.The carrier that cuts and the insertion fragment of sudden change are mixed with different ratios and with the T4 ligase enzyme 15 ℃ of processing whole night.Purifying connects product and with electroporation it is transformed into the e. coli host bacteria strain.
The aliquot sample of expressing culture is covered penbritin to be selected on the substratum so that be determined at the number of single transformant in each conversion.Under the situation that has glycerine as cryoprotectant, the conversion that has maximum single transformant under each induced mutation rate is stored in-70 to-80 ℃.
Then each library is coated on the large size penbritin selectivity agar plate.(QPix2, Genetix Ltd) changes single bacterium colony in 384 orifice plates that comprise the 2X Luria meat soup that contains penbritin and 10%w/v glycerine over to automatic bacterium colony pickout apparatus.These flat boards are hatched whole night in 30 ℃, allow the culture growth, are stored in-70 to-80 ℃ then.It is enough low allowing the culture growth to be added to amounts of glycerol in the 2X Luria meat soup, however enough height so that cryoprotection to be provided.Some mutagenesis (the Mg that are used for using have subsequently been prepared in this way + 2) thousands of bacterium colonies under the level.
The extract library prepares part 1---fermentation: the corresponding library that is suitable for screening the partial purification extract of purpose from the preparation of above-mentioned clone library.The first step of this program is that each clone's of preparation small-scale is expressed culture.These cultures are grown with 96 well format; Therefore library, every 384 hole flat board has 4 to express culture plate.Shift the hole of 1 μ l to the 96 hole seed flat boards that comprise 150 μ l culture medium A (referring to following table 3) from each hole of clone library flat board.The seed flat board vibrates whole night at 1150rpm under 30 ℃ in the dull and stereotyped couveuse/vibrator of iEMS (ThermoElectron).These inoculums are used to inoculate identical substratum then, go up inoculation 10 μ l at large size 96 orifice plates (Nunc#267334) specifically and go in the 300 μ l culture medium A.These flat boards are hatched whole night in 37 ℃.Expression plasmid comprises transcriptional regulatory element, and it is allowed at 37 ℃ and expresses and can not be 30 ℃ of expression.After hatching whole night, culture is typically with the 1-10% cloning by expression protein of total cell protein matter.By centrifugal from these cultures harvested cell.These cells are by frozen (20 ℃) or processing immediately as described below.
Table 3. culture medium A (filtration sterilization before using)
Composition Concentration
MgSO 4.7H 2O 0.2g/L
Citric acid .H 2O 2g/L
K 2HPO 4 10g/L
NaNH 4PO 4.4H 2O 3.5g/L
MgSO 4 2mM
Casamino acids 2.5g/L
Glucose 2g/L
VitB1 .HCl 10mg/L
Penbritin 100mg/L
The extract library prepares part 2---extracts: from the cell mass of fermentation step resuspension and change 384 hole thermal cycler flat boards in 30 μ l lysis buffers (following table 4).Notice that this damping fluid comprises N,O-Diacetylmuramidase to help lysis and two kinds of nucleases to remove RNA and DNA from extract.Make flat board stand 3 round-robin freeze-thaws (70 ℃ freezing, and 37 ℃ thaw, and each step is no less than 15 minutes) with lysing cell.Add ammonium sulfate (the 0.75M solution of 5 μ l) and hatched flat board 15 minutes so that precipitation and inactivation contaminating protein matter comprise the nuclease that external source is added at 75 ℃.Flat board changed supernatant liquor over to new 384 hole thermal cycler flat boards in centrifugal 15 minutes then in 3000 * g.These extract flat boards are frozen to be used for screening subsequently in-20 ℃.Every hole comprises the mutant library polysaccharase of about 0.5-3 μ M.
Table 4. lysis buffer
Composition Concentration or per-cent
Tris pH 8.0 20mM
EDTA 1mM
MgCl 2 5mM
TLCK 1mM
Leupeptin 1μg/ml
PefabloC 0.5mg/ml
Tween
20 0.5％v/v
N,O-Diacetylmuramidase (powder) 2mg/ml
RNase 0.025mg/ml
DNase I 0.075 unit/μ l
Extension speed screening extract library for improvement: M13mp18 single stranded DNA (M13; GenBank accession number X02513), apparatus has the oligonucleotide of following sequence to cause: 5 '-GGGAAGGGCGATCGGTGCGGGCCTCTTCGC-3 ' (SEQ ID NO:72)
Template molecule as the elongation test screening.0.5-1.0 μ l extract is added in the reaction mother liquor that 10-20 μ l on the 384 hole PCR flat boards comprises the M13 template that 0.5-1nM caused.The extension of the template that has caused detects with CCD photographic camera every 10-30 in improved dynamic thermal cycler second (sees Watson, above).Typical reaction mother liquor is listed in as follows.Mother liquor always comprises metal ion, the magnesium of 1-4mM normally, the mixture of whole 4 kinds of dNTPs or dNTP analogue, the buffer reagent composition of control pH and ionic strength, the typical case is 25mM Hepes (Tricine) pH 8.3/35mM KOAc, and 0.6 * SYBR Green I (molecular probe), it allows the fluoroscopic examination that primer strand extends.To extend deutero-fluorescence in order from background fluorescence, distinguishing, to comprise in the experiment that primer strand extends the parallel hole that is prevented from, for example, by adding such as the metal chelator of EDTA or from reaction mother liquor, removing Nucleotide.
To have the mutant enzyme that improved nucleic acids acids is extended speed under the situation of ribonucleotide in order finding having, to use aforesaid method in the extension speed that has and lack operation extension under the situation of ribonucleotide and relatively produce.Add the extension speed of high-caliber ribonucleotide (for example 50: 50 mixtures of rATP and dATP) reduction parent enzyme G46E L329A E678G CS5.In this screening, identify the mutant extract of the ribonucleotide inhibition level that represents reduction.On the scale of thousands of extracts, carry out preliminary screening.Select the extract of the highest some per-cents to be used for screening again.Be sampled to fresh growth medium and regrow corresponding to the culture hole of the highest extract and comprise all the highest producers' new culture plate with generation, and the culture that is used for some parents of comparison.Then these culture plates are sent into identical screening procedure, to obtain more data about candidate's mutant.This second take turns screening after, relatively small number purpose extract seems still as one man to demonstrate the extension speed with respect to parent clone's improvement.These clones are selected for further test.It is pure with what guarantee to clone at first they to be rule on the selectivity agar plate, and the dna sequence dna to the zone of pol gene sudden change checks order to determine to be present in any single clone's sudden change then.With this work concurrently, in shake-flask culture, produce competent mutant enzyme, measure its concentration to use based on the light densitometry of gel behind the mode partial purification that is similar to the preliminary extract of preparation.These quantitative extracts are compared with the parent enzyme that equates protein concn under the condition that is used to screen.Should final screening guarantee that observed difference was not simple protein concn effect.
In the end one take turns after the screening, seem still to have the extension speed of improvement under the situation that 4 are cloned in ribonucleotide.Determine the sequence of coding with respect to these 4 clones of the following amino acid variation of parent strain:
Clone 1:S553T D640G D664G E830A
Clone 2:S671F
Clone 3:F557L I669F
Clone 4:Q601R Y739C V749A
For clone 2, clearly the S671F sudden change must determine observed phenotype, because it is the unique amino acid mutation among this clone.For other 3 clones, originally can not conclude which sudden change, or the combination of sudden change is determining observed phenotype.Therefore, use the restriction fragment exchange DNA and parental plasmid's combination, thereby make independent sudden change separated from one another from the mutant plasmid.This is to realize easily under the situation of the restriction site of wanting to exist between the isolating sudden change carrier uniqueness.For clone 1, above-mentioned site is present between whole 4 sudden changes, other plasmids that correspondingly may prepare the plasmid that comprises each sudden change respectively and carry any 2 or 3 in 4 original sudden changes.For clone 4, between Y739C and V749A, do not have above-mentioned site, but a site is arranged between Q601R and Y739C.Therefore may prepare that coding only carries the plasmid DNA of polysaccharase of Q601R sudden change and another carries the plasmid of Y739C/V749A combination.
These new plasmids are transformed into escherichia coli host, and express, be purified to homogeneity and quantitative poly protein.The new mutant enzyme of these generations is compared with the primary mutant enzyme with parental type under original screening conditions.Be clear that very that from these data sudden change D640G determines the phenotype of mutant clone 1 improvement individually, sudden change I669F is determining the improvement in the mutant clone 3, and sudden change Q601R is determining the improvement in the mutant clone 4.
Then these active sudden changes are bonded to each other, and move into different CS type main chains (referring to, for example, table 2 above), as mentioned above, exchange with restriction fragment once more and create the expression plasmid that needs, then plasmid is transformed into escherichia coli host, express, be purified to homogeneity and quantitative mutant polysaccharase at last.The ability of extending the M13 DNA that has caused at the mutant that these new combinations of test are arranged under the situation of ribonucleotide.Enjoyably, discovery combinatorial mutagenesis D640G, S671F and Q601R increase with respect to the extension speed that the clone who only carries single sudden change produces.The double combination mutant of test comprises D640G S671F and Q601R S671F, also demonstrates the extension speed with respect to the improvement of the bacterial strain that only carries single sudden change.In addition, combination mutant has also proved with parental type and has compared, the extension speed of the improvement when only having dNTPs on the M13 DNA that has caused, and also observe when extending the speed experiment when under low enzyme concn or high relatively salt concn, carrying out, with respect to the improvement degree maximum of parental type.These observationss are repeated when comprising that not ribose mixes the hereditary main chain of sudden change (riboincorporating mutation) E678G when combinatorial mutagenesis is moved on to.Surprisingly, even under the E678 background, independent mutant enzyme and combinatorial mutagenesis type enzyme even than their corresponding E678 " parent " " faster ".These and other feature of mutant polysaccharase of the present invention is further illustrated in the following example.
Example II: the salt concn that G46E L329A E678G CS5 mutant DNA polymerases is changing Under character
The nucleic acid of the various mutant of G46E L329A E678G CS5 archaeal dna polymerase extends speed and measures under the situation that 90% ribose adenosine triphosphate (ribo ATP or rATP) arranged.Reaction mixture comprises 25mM Hepes pH 8.3,20mM (Fig. 4) or 60mM (Fig. 5) KOAc, 3mM MgCl 2, 2.5%v/v storage damping fluid (50%v/v glycerine, 100mMKCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), 1%DMSO, 1 * SYBR Green I, the M13 that 0.5nM has caused and 5nM enzyme.Nucleotide adds with the final concentration of 0.1mM dGTP, 0.1mM dTTP, 0.1mM dCTP, 0.01mM dATP and 0.09mM ribo ATP.The parallel reaction that does not comprise Nucleotide also is established.Total overall reaction runs on the 384 hole thermal cycler flat boards in quadruplicate with 20 μ l volumes.Fluoroscopic examination is used in extending in the dynamic thermal cycler in 64 ℃ of the M13 template that has caused down, obtains reading in per 10 seconds.Calculate same reaction repeated experiments mean number and deduct parallel negative Nucleotide reaction.From the data that produce, extend speed with the linear regression analysis estimation.
Point out that as top Figure 4 and 5 show the result who obtains from these are analyzed.For example, Figure 4 and 5 are illustrated when ribonucleotide is present in reaction mixture and be incorporated on the dna profiling, and improved nucleic acids acids is extended speed and come from various mutant described herein.Further show, for example, when some sudden change is combined in the single mutant enzyme, even observe further extension rate improvement.
EXAMPLE III: the property of G46E L329A CS5 dna polymerase mutant body under the salt concn that changes Matter
The various mutant of G46E L329A CS5 archaeal dna polymerase, and dwell hot bacterial classification Z05DNA polysaccharase and truncation type deltaZ05 archaeal dna polymerase thereof (referring to, for example, authorize the U.S. Patent number 5 of Abramson etc. " Mutated thermostable nucleic acidpolymerase enzyme from Thermus species Z05 " by name October 3 nineteen ninety-five, 455, authorized the U.S. Patent number 5 of Abramson etc. " DNA encoding thermostablenucleic acid polymerase enzyme from thermus species Z05 " by name on October 7th, 170 and 1997,674,738) it is determined that nucleic acid extends speed.Reaction mixture comprises 25mM Hepes pH 8.3,0mM (Fig. 6) or 60mM (Fig. 7) KOAc, 3mM MgCl 2, 2.5%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), 1%DMSO, the M13 that 1 * SYBR GreenI, 0.5nM have caused and 5nM enzyme.Nucleotide adds with the final concentration of 0.1mM dGTP, 0.1mMdTTP, 0.1mM dCTP and 0.1mM dATP.The parallel reaction that does not comprise Nucleotide also is established.Total overall reaction runs on the 384 hole thermal cycler flat boards in quadruplicate with 20 μ l volumes.Fluoroscopic examination is used in extending in the dynamic thermal cycler in 64 ℃ of the M13 template that has caused down, obtains reading in per 10 seconds.Calculate same reaction repeated experiments mean number and deduct parallel negative Nucleotide reaction.From the data that produce, extend speed with the linear regression analysis estimation.
Be shown in the data interpretation of Fig. 6 and 7, for example, even be not present in reaction mixture when ribonucleotide, and even comprising that not ribonucleotide mixes in the hereditary main chain of sudden change E678G, some sudden change described herein causes that improved nucleic acids acids extends speed.Further show, for example, this rate improvement even bigger when sudden change is made up in single mutant enzyme.
EXAMPLE IV: salt concn is to the effect of the extension speed of various mutant CS5 archaeal dna polymerases
The various mutant of G46E L329A CS5 archaeal dna polymerase, and the nucleic acid of dwell hot bacterial classification Z05DNA polysaccharase and truncation type deltaZ05 archaeal dna polymerase thereof extension speed is determined.Reaction mixture comprises 25mM Hepes pH 8.3,0-100mM KOAc, 3mM MgCl 2, 2.5%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mMTris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), 1%DMSO, 1 * SYBR Green I, the M13 that 0.5nM has caused, and the enzyme of 25nM (Fig. 8) or 5nM (Fig. 9).Nucleotide adds with the final concentration of 0.1mM dGTP, 0.1mM dTTP, 0.1mM dCTP and 0.1mM dATP.The parallel reaction that does not comprise Nucleotide also is established.Total overall reaction runs on the 384 hole thermal cycler flat boards in quadruplicate with 20 μ l volumes.Fluoroscopic examination is used in extending in the dynamic thermal cycler in 64 ℃ of the M13 template that has caused down, obtains reading in per 10 seconds.Calculate same reaction repeated experiments mean number and deduct parallel negative Nucleotide reaction.From the data that produce, extend speed with the linear regression analysis estimation.
Being shown in Fig. 6 illustrates together with other character with 7 data, for example, the nucleic acid of the increase that some mutant described herein gives extends speed and keeps in very wide salt and enzyme concn scope, and these sudden changes give the extension that increases speed in comprising the genetic background of complete proofreading activity.
EXAMPLE V: the use of various mutant CS5 archaeal dna polymerases in RT-PCR
Based on Mg 2+RT: Mg is being arranged + 2Situation under assessment independent with combination sudden change Q601R, D640G and S671F to the influence of PCR and RT-PCR efficient.Reaction all comprises following ingredients: 50mM Hepes pH 8.0,2.5mM Mg (OAc) 2, 6%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mMEDTA, 1mM DTT, 0.2%Tween 20), 0.2 * SYBR Green I, 0.02 unit/μ lUNG, dATP, each 0.2mM of dCTP and dGTP, dUTP 0.3mM, dTTP 0.03mM, and every kind of primer 2 00nM, wherein primer 3 '-end comprises 2 '-amino-C.
Enzyme uses by concentration and KOAc optimum condition that they are measured in advance.These provide in table 5.
Table 5.
Polysaccharase Pol(nM) KOAc(mM) Polysaccharase Pol(nM) KOAc(mM)
G 236 25 GL 236 25
GD 59 50 GLD 59 50
GS 118 25 GLS 118 25
GDS 23.6 25 GLDS 23.6 25
GQDS 23.6 100 GLQDS 23.6 100
Every kind of enzyme is all with 10 6The dna profiling (pAW109 plasmid DNA) and 10 of copy/50 μ l reaction 6The RNA template (pAW109 transcript) of copy/50 μ l reaction is tested together.Be reflected in the dynamic thermal cycler (ABI 5700 thermalcycler) and carry out.Thermal circulation parameters is: 50 ℃ 2 minutes; 65 ℃ 45 minutes; 93 ℃ 1 minute; 40 round-robin then: 93 ℃ of 15 seconds and 65 ℃ 30 seconds.The analysis of fluorescence data are to measure Ct value (in the appearance of the above fluorescence of baseline) (Figure 10).More particularly, the data (also referring to Figure 11) that are shown in Figure 10 are illustrated together together with other character, and for example, with respect to corresponding parent or not mutated type enzyme, sudden change described herein independent or combination has improved the Mg of mutant enzyme 2+The active efficient of activatory reverse transcription.For example, when the time that reverse transcription allows foreshortened to 5 minutes, the performance of GLDS enzyme was good, shown in Figure 11 (relate to following other).
The RT time shorten based on Mg 2+RT: use the RT time of 45 minutes or 5 minutes, Mg arranged + 2Situation under assessment independent with combination sudden change Q601R, D640G and S671F to the influence of RT-PCR efficient.Reaction all comprises following ingredients: 50mM Hepes pH 8.0,2.5mM Mg (OAc) 2, 6%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.2%Tween20), 1%DMSO, 0.2 * SYBR Green I, 0.02 unit/μ l UNG, each 0.2mM of dATP, dCTP and dGTP, dUTP 0.3mM, dTTP 0.03mM, and every kind of primer 2 00nM, wherein primer 3 '-end comprises 2 '-amino-C.
Enzyme uses by concentration and KOAc optimum condition that they are measured in advance.These provide in table 6 and 7:
The table 6.45 minute RT time:
Polysaccharase Pol(nM) KOAc(mM) Polysaccharase Pol(nM) KOAc(mM)
G 236 25 GL 236 25
GD 59 50 GLD 59 50
GS 118 25 GLS 118 25
GDS 23.6 25 GLDS 23.6 25
GQDS 23.6 100 GLQDS 23.6 100
The table 7.5 minute RT time:
Polysaccharase Pol(nM) KOAc(mM) Polysaccharase Pol(nM) KOAc(mM)
G 118 25 GL 236 55
GD ～ ～ GLD 94.4 50
GS 118 25 GLS 118 25
GDS 23.6 25 GLDS 106.2 50
GQDS ～ ～ GLQDS 23.6 100
This condition of～expression is not carried out
Every kind of enzyme is all with 10 6The RNA template (pAW109 transcript) of copy/50 μ l reaction is tested together.Be reflected in the dynamic thermal cycler (ABI 5700) and carry out.Thermal circulation parameters is: 50 ℃ 2 minutes; 65 ℃ 5 minutes or 45 minutes; 93 ℃ 1 minute; 40 round-robin then: 93 ℃ of 15 seconds and 65 ℃ 30 seconds.The analysis of fluorescence data are to measure Ct value (in the appearance of the above fluorescence of baseline) (Figure 11).
The RT time shorten based on Mn 2+RT: use the RT time of 45 minutes or 5 minutes, Mn arranged + 2Situation under assessment independent with combination sudden change Q601R, D640G and S671F to the influence of RT-PCR efficient.Reaction all comprises following ingredients: 50mM Hepes pH 8.0,1mM Mn (OAc) 2, 6%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.2%Tween20), 1%DMSO, 0.2 * SYBR Green I, 0.02 unit/μ l UNG, each 0.2mM of dATP, dCTP and dGTP, dUTP 0.3mM, dTTP 0.03mM, and every kind of primer 2 00nM, wherein primer 3 '-end comprises 2 '-amino-C.
Enzyme uses by concentration and KOAc optimum condition that they are measured in advance.These provide in table 8 and 9:
The table 8.45 minute RT time:
Polysaccharase Pol(nM) KOAc(mM) Polysaccharase Pol(nM) KOAc(mM)
G 236 55 GL 236 55
GD ～ ～ GLD 59 55
GS 118 55 GLS 118 55
GDS 23.6 55 GLDS 23.6 70
GQDS 23.6 100 GLQDS 23.6 100
The table 9.5 minute RT time:
Polysaccharase Pol(nM) KOAc(mM) Polysaccharase Pol(nM) KOAc(mM)
G ～ ～ GL 354 68
GD ～ ～ GLD ～ ～
GS ～ ～ GLS 59 55
GDS ～ ～ GLDS 23.6 70
GQDS 59 100 GLQDS 11.8 100
This condition of～expression is not carried out
Every kind of enzyme is all with 10 5The RNA template (pAW109 transcript) of copy/50 μ l reaction is tested together.Be reflected in the dynamic thermal cycler (ABI 5700) and carry out.Thermal circulation parameters is: 50 ℃ 2 minutes; 65 ℃ 5 minutes or 45 minutes; 93 ℃ 1 minute; 40 round-robin then: 93 ℃ of 15 seconds and 65 ℃ 30 seconds.The analysis of fluorescence data are to measure Ct value (in the appearance of the above fluorescence of baseline) (Figure 12).More particularly, the data that are shown in Figure 12 are illustrated together together with other character, for example, and the Mn of improvement 2+Activatory reverse transcription efficient is caused by described herein specific sudden change independent or combination, and this improvement strengthens when the time that is provided for reverse transcription shortens.
Example VI: use low-level ribonucleotide triphosphate to mix the fracture of generation
Making the fracture of PCR product is useful sometimes, for example when assay products in based on the test of hybridization.If ribonucleotide has mixed the PCR product, fracture can be finished easily with alkali and thermal treatment.Replace the fragment that is enough to obtain optimal length for the low-level relatively ribose of above-mentioned application.The ability of the PCR product that the ribose that various mutant DNA polymerases generation length are 1kb replaces proves in the following embodiments.
Reaction mixture is by 100mM Hepes pH 8.3,75mM KOAc, 5%v/v glycerine, 2.5mM Mg (OAc) 2, the 50nM enzyme, 0.1%v/v DMSO and 2.5%v/v enzyme storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mMEDTA, 1mM DTT, 0.5%Tween 20) form.The various mixtures of dNTPs and rNTPS have been tested.In all cases, the total amount of rATP and dATP is 200 μ M, and the total amount of dCTP and rCTP and dGTP and rGTP also is.The total amount of dTTP and rTTP is 40 μ M, and the total amount of dUTP and rUTP is 360 μ M.The whole 4 kinds of rNTPS of common interpolation are up to 10% (referring to " rNTP series " (%rNTP indicates) of Figure 13 A and B) of sum above the corresponding swimming lane of gel in this analysis, perhaps add 50% (referring to " rATP series " (%rATP indicates above the corresponding swimming lane of gel) of Figure 13 A and B) that rATP is up to sum separately.The test enzyme be GQDSE, CS6-GQDSE, GLQDSE, GDSE, GLDSE, GLDE, GE and GL and GLE 4: 1 mixtures (G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, E=E678G).
Reaction mixture comprises the primer that is used for producing from the M13 template 1kb product.Primer uses with every kind of 200nM, wherein said primer 3 '-end comprises 2 '-amino-C.Per 100 μ l reaction solutions add 10 6The M13 DNA of copy.
Be reflected in ABI 9700 thermal cyclers and move.Thermal circulation parameters be 50 ℃ 15 seconds; 92 ℃ 1 minute; 30 round-robin then: 92 ℃ of 15 seconds and 62 ℃ of extension steps of 4 minutes subsequently.The ability of preparation total length amplicon is measured by agarose gel electrophoresis under the various conditions of test, and the egel-48 2% (invitrogen) goes up the every kind of reaction solution (Figure 13 A and 13B) of sample 5 μ l on every swimming lane.More particularly, these figure show that for example, some mutant enzyme described herein exists can production total length (1kb) amplicon than under corresponding parent or the higher levels of ribonucleotide of not mutated type G46E CS5R enzyme in reaction mixture.For example, the mixture of GL CS5 and GLE enzyme prepares amplicon under the ribonucleotide of the highest level of this embodiment test, but because GL CS5 polysaccharase can not mix ribonucleotide, it is relatively low to contain the ribonucleotide level of mixing amplicon in these amplicons.
Making the fracture of these amplicons by following then: 2 μ l amplicons dilute 27.5 in 0.3N NaOH and 20mMEDTA *, then in 98 ℃ of heating 10 minutes.By adding among the 2.5 μ l 6N HCl and the amplicon of fracture.Be the breaking degree that mensuration reaches, before and after fracture, use the quantitative PCR that does not contain UNG to compare the copy number of the interior segments of amplicon.Because the observed circulation delay of fracture is the index of breaking degree (and ribonucleotide mixes).The ribonucleotide that increases mixes and causes Ct to postpone to increase.For this amplification, reaction mixture is by 100mM Hepes pH 8.3,50mM KOAc, 5%v/v glycerine, 2.5mM Mg (OAc) 2, 20nM GQDS, 0.5%DMSO, 0.1 * SYBR Green I, 2.5%v/v enzyme storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), each 200 μ M of dCTP, dGTP and dATP, dUTP 360 μ M and dTTP40 μ M form.Reaction mixture is used for producing from amplicon fracture and not fracture the product of 340bp, is used to the diluent that ruptures 10,000 times of these templates of dilution more certainly.Primer sequence uses with every kind of 200nM, wherein said primer 3 '-end comprises 2 '-amino-C.
Be reflected in the dynamic thermal cycler and run on 384 orifice plates with every reaction 20 μ l.Thermal circulation parameters is: 50 ℃ 15 seconds; 92 ℃ 1 minute; 46 round-robin then: 92 ℃ of 15 seconds and 62 ℃ of extension steps of 1 minute subsequently.Mensuration threshold Ct and Ct relatively more corresponding fracture and that do not rupture, thus delta Ct produced for the rNTP condition of each enzyme/test.In this embodiment, the quantity (reflection improvement the ability of mixing NTPs under the situation of dNTPs is being arranged) of mixing NTP is big more, and the Ct behind deltaCt or the alkali inductive cleavage postpones big more.These are shown in Figure 14 A and 14B.Data presentation, for example, mutant enzyme of the present invention is superior aspect generation has mixing of ATP in the PCR product that the ribonucleotide of raising degree replaces or NTP.Any exemplary enzyme and parent's mixture " GL/GLE " or with " C5R " relatively.The fracture that increases derives from the ability of mixing the ribonucleotide of limiting concentration in the presence of deoxynucleotide that the ribonucleotide of increase mixes and improves.
Cross experiment often relates to the molecule that vitamin H is connected to detection.Therefore mixing vitamin H into, the PCR product is useful.If vitamin H is connected to ribonucleotide, each fragment (remove the fragment of 3 ' least significant end, therefore it usually and other primer is complementary can not provide information) will be carried single biotin moiety, and this will cause each fragment to produce equal signal.
Measure ribonucleotide that various enzymes will be connected with vitamin H and mixed ability in the PCR product into, as described below.Reaction mixture is by 100mM Hepes pH 8.3,75mMKOAc, 5%v/v glycerine, 2.5mM Mg (OAc) 2, 50nM enzyme, 0.1%DMSO, 2.5%v/v enzyme storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), each 200 μ M of dCTP+ analogue, dGTP and dATP, dUTP 360 μ M, and dTTP 40 μ M form.Test the rCTP that is up to total amount 40% or be up to the vitamin H-LC-rCTP of total amount 50%.The test enzyme (CS5 polysaccharase) be GE, GQDSE, GDSE and GL and GLE 4: 1 mixtures (G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, E=E678G).
Reaction mixture be used for by comprise 2 '-every kind of 200nM primer sequence of amino-C produces the 1kb product from the M13 template.Add 5 * 10 in per 50 μ l reaction solutions 5Copy M13 DNA.Be reflected in ABI 9700 thermal cyclers and move.Thermal circulation parameters be 50 ℃ 15 seconds; 92 ℃ 1 minute; 30 round-robin then: 92 ℃ of 15 seconds and 62 ℃ of extension steps of 4 minutes subsequently.The ability that produces the total length amplicon under various test conditions is measured by agarose gel electrophoresis, goes up sample 5 μ l (Figure 15 A and B) on every kind of reaction solution of every swimming lane at 2%egel-48 (Invitrogen).More particularly, Figure 15 A and B show that for example, mutant GQDSE and GDSE can both produce amplicon than corresponding parent or not mutated type G46E CS5R enzyme in higher levels of rCTP and biotinylated rCTP.Though further the GL/GLE mixture can produce amplicon, these amplicons will have low-level rCTP or biotinylated rCTP mixes, because the GL enzyme can not mix these compounds.
Making the fracture of these amplicons by following then: 2 μ l amplicons dilute 27.5 in 0.3N NaOH and 20mMEDTA *, then in 98 ℃ of heating 10 minutes.By adding among the 2.5 μ l 6N HCl and the amplicon of fracture.Be the breaking degree that mensuration reaches, before and after fracture, use the quantitative PCR that does not contain UNG to compare the copy number of the interior segments of amplicon.Because the observed circulation delay of fracture is the index of breaking degree (and ribonucleotide mixes).Thereby the ribonucleotide that increases mixes and causes Ct to postpone to increase.For this amplification, reaction mixture is by 100mM Hepes pH 8.3,50mM KOAc, 5%v/v glycerine, 2.5mM Mg (OAc) 2, 20nMGQDS, 0.5%DMSO, 0.1 * SYBR Green I, 2.5%v/v enzyme storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), each 200 μ M of dCTP, dGTP and dATP, dUTP 360 μ M and dTTP 40 μ M form.Reaction mixture is used for producing from amplicon fracture and not fracture the product of 340bp, is used to the diluent that ruptures 10,000 times of these templates of dilution more certainly.Primer sequence uses with every kind of 200nM, wherein each primer all comprise 2 '-amino-C.
Be reflected in the dynamic thermal cycler and run on 384 orifice plates with every reaction 20 μ l.Thermal circulation parameters is: 50 ℃ 15 seconds; 92 ℃ 1 minute; 46 round-robin then: 92 ℃ of 15 seconds and 62 ℃ of extension steps of 1 minute subsequently.Mensuration threshold Ct and Ct relatively more corresponding fracture and that do not rupture, thus delta Ct produced for the rNTP condition of each enzyme/test.These are shown in Figure 16 A and 16B.More particularly, Figure 16 A and 16B illustrate, and for example, can increase breaking degree with mutant enzyme when using rCTP or biotinylated rCTP, have the amplicon that higher levels of ribonucleotide mixes because they can produce than corresponding parent enzyme.
Example VII A: pyrophosphorolysis activatory polymerization
Compare G46E L329A E678G CS5 archaeal dna polymerase and G46E L329A D640S671F E678G CS5 archaeal dna polymerase and carried out the ability of pyrophosphorolysis activatory polymerization (" PAP ").Reaction buffer is by 100mM Hepes pH 8.0,2.5-50mMG46E L329A E678G CS5 archaeal dna polymerase or 2.5-50mM G46E L329A D640GS671F E678G CS5 archaeal dna polymerase, 50nM KOAc, 10%v/v glycerine, 0.04U/ μ lUNG, 4mM Mg (OAc) 2, 1%DMSO, 0.2 * SYBR Green I, 2.5%v/v enzyme storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mMEDTA, 1mM DTT, 0.5%Tween 20), each 0.2mM of dATP, dCTP and dGTP, dUTP 0.4mM and 100 μ M pyrophosphate salts are formed.Intersection titration M13 template and enzyme.The M13 concentration of using is per 20 μ l reaction solutions 0,10 4, 10 5With 10 6Copy.The enzyme concn of using is 2.5nM, 5nM, 10nM, 15nM, 20nM, 25nM, 35nM and 50nM.Be reflected in the 384 hole thermal cyclers in triplicate and be provided with, use following loop parameter: 50 ℃ 2 minutes; 90 ℃ 1 minute; 46 round-robin then: 90 ℃ of 15 seconds and elongating temperatures 60 seconds of 62 ℃ subsequently.
Article one, primer 3 '-end comprises 2 '-amino-C, another primer 3 '-end comprises 2 '-PO 4-A (promptly 2 '-terminating nucleotide).These primers that add reaction mixture to every kind 0.1 μ M will produce the product of 348bp from the M13 template.Yet, 2 of second primer 3 ' end '-PO 4-A residue produces the terminator effect effectively.In order to be used as primer, it must be excised the terminal residue activation by pyrophosphorolysis.
The analysis of fluorescence data are to measure elbow value (C (t)) (appearance of fluorescence on baseline).C (t) value of G46E L329A E678G CS5 archaeal dna polymerase is shown in Figure 17.C (t) value of G46E L329AD640G S671F E678G CS5 archaeal dna polymerase is shown in Figure 18.Further, Figure 17 and 18 shows, for example, uses mutant enzyme to cause more effective PAP-PCR than corresponding not mutated type or parent enzyme under lower enzyme concn.Gel analysis shows that the amplicon among this embodiment in the no template reaction thing may be the specific product that derives from the M13 of environment.
Example VII A I: the influence of speed is extended in the sudden change of selection to the hot bacterial classification Z05 archaeal dna polymerase of dwelling
Some sudden changes of the screening and separating of describing by example I changed over to the hot bacterial classification Z05 archaeal dna polymerase of dwelling (referring to, for example, authorize the U.S. Patent number 5 of Abramson etc. " MUTATED THERMOSTABLE NUCLEIC ACID POLYMERASEENZYME FROM THERMUS SPECIES Z05 " by name October 3 nineteen ninety-five, 455, authorized the U.S. Patent number 5 of Abramson etc. " DNA ENCODINGTHERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROMTHERMUS SPECIES Z05 " by name on October 7th, 170 and 1997,674,738).At first, measure by the comparison that use is shown in Fig. 1 corresponding to the amino acid sites of these sudden changes.Following name is pressed in these sudden changes: " Q "=T541R; " D "=D580G; And " S "=A610F.By use be called as overlapping extension PCR (overlap extension PCR) (referring to, for example, Higuchi, R.in PCRProtocols:A Guide to Methods and Applications, ed.Innis, Gelfand, Sninsky and White, Academic Press, 1990 and Silver etc., " Site-specificMutagenesis Using the Polymerase Chain Reaction ", in " PCR Strategies ", ed.Innis, Gelfand, and Sninsky, Academic Press, 1995) method is introduced these sudden changes the plasmid of coding Z05 archaeal dna polymerases.In the method, at first produce two amplicons, both are respectively at the upstream and downstream in mutagenesis site, and a primer of each reaction is introduced in described sudden change.Use the non-mutagenic primer combination in the outside and these amplified productions that increase again then.The amplicon that produces comprises the sudden change of introducing and is designed to restriction site across the carrier uniqueness, therefore can be used for amplicons cloned is advanced vector plasmid DNA.For the ease of from the clone of generation of the mixture that may comprise mutant and wild-type clone, selecting the sudden change of needs, can introduce into mutagenic primer to the diagnostic restriction site as required.This program may be introduced the unwanted sudden change that is caused by low fidelity PCR, therefore is necessary the clone who produces is checked order to confirm only to have produced the sudden change that needs.Be proved in case suddenly change, make up with their combinations with one another or with previous isolating E683R sudden change (ES112) (equaling the Application No. 20020012970 of " the High temperature reverse transcription usingmutant DNA polymerases " by name of submission on March 30 calendar year 2001 referring to Smith) according to previously described restriction fragment exchange.
The expression plasmid that produces with this method is used to prepare according to the description in the example I before the protein purification of various mutant.The nucleic acid of measuring various mutant then extends speed.Reaction mixture comprises 25mM Hepes pH 8.3,100mM KOAc, 3mM MgCl 2, 2.5%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), 1%DMSO, the M13 that 1 * SYBR GreenI, 0.5nM have caused and 5nM enzyme.Nucleotide adds with the final concentration of 0.1mM dGTP, 0.1mMdTTP, 0.1mM dCTP and 0.1mM dATP.The parallel reaction that does not comprise Nucleotide also is established.Total overall reaction runs on the 384 hole thermal cycler flat boards in quadruplicate with 20 μ l volumes.Fluoroscopic examination is used in extending in the dynamic thermal cycler in 64 ℃ of the M13 template that has caused down, obtains reading in per 10 seconds.Calculate same reaction repeated experiments mean number and deduct parallel negative Nucleotide reaction.From the data that produce, extend speed (referring to Figure 19) with the linear regression analysis estimation.These data show that for example, sudden change described herein in some cases also has useful effect in the background of non-chimeric Thermus archaeal dna polymerase.
The titration of example I X:HIV dna profiling
Genomic dna exist with non-existent situation under carry out the relevant HIV dna profiling titration of PAP.Figure 20 is presented at the gel photograph that the PCR product detects under the reaction conditions that changes in this analysis.These data show, for example, use sealing primer described herein can obtain specific amplification and sensitivity with respect to the improvement of the reaction of not using those primers.
More particularly, reaction is to use ABI 5700 sequence detection systems to carry out with following temperature distribution:
50 ℃ 2 minutes
93 ℃ 1 minute
93 ℃, 15 seconds → 52 ℃, 4 minutes * 4 circulations
90 ℃, 15 seconds → 55 ℃, 4 minutes * 56 circulations
Following reaction conditions is total by being responded:
Mother liquor composition Concentration
Hepes (pH 8.0) 100mM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
Primer
3 or primer 1 200nM
Primer
4 or primer 2 200nM
KOAc 110mM
SYBR Green I 0.2X
NaPPi 225μM
Mg(OAc) 2 2.5mM
Tth stores damping fluid (0.2%Tween) 6％v/v
GLQDSE CS5 archaeal dna polymerase 10nM
Notice that " GLQDSE CS5 archaeal dna polymerase " refers to G46E L329A Q601R D640GS671F E678G CS5 archaeal dna polymerase.Notice that further " Tth stores damping fluid " comprises 0.2%Tween 20,20mM Tris pH8.0,0.1mM EDTA, 100mM KCl, 1mM DTT and 50%v/v glycerine.In addition, each reaction volume is added to 50 μ l with diethylpyrocarbonate (DEPC) treated water.
The reacted constituent that changes comprises non-sealing primer (reaction of " non-sealing primer " expression among Figure 20) and with 2 '-primer of phosphoric acid-U sealing (promptly comprise in 2 ' position 2 of phosphate group '-terminating nucleotide, the reaction of representing referring to " primer of sealing " among Figure 20).Reaction also comprises (referring to the reaction of " 25ng genomic dna " expression among Figure 20) or lacks the 25ng human genome DNA that (referring to the reaction of " clean target " expression among Figure 20) joins mixture.As what further show among Figure 20, reaction also comprises 10 5, 10 4, 10 3, 10 2Or 10 1The linearization plasmid DNA that comprises target nucleic acid of copy, it is at 1 μ l HIV diluents (10mM Tris, 0.1mM EDTA, 20 μ g/mL Poly A, and 0.09%NaN 3) middle dilution, perhaps in " negative (Neg) " reaction, also comprise 1 μ l HIV diluents.Specified primer is to the product from plasmid DNA amplification 170bp.
Embodiment X: the amplification of mutant K-ras plasmid template in wild-type K-ras plasmid template background
The mutant K-Ras plasmid template of the various copy numbers of amplification in wild-type K-Ras plasmid template background, and relatively seal and non-sealing primer.Figure 21 shows for the observed threshold circulation of the various mutant K-Ras plasmid template copy numbers (X-axis) that use in these reactions (C T) value (y axle) chart.Figure 21 further illustrates, for example, and the resolving power that uses sealing primer described herein to obtain to improve.
Reaction is to use ABI 5700 sequence detection systems to carry out with following temperature distribution:
50 ℃ 2 minutes
93 ℃ 1 minute
92 ℃, 15 seconds → 65 ℃, 2 minutes * 60 circulations
Following reaction conditions is total by being responded:
Mother liquor composition Concentration
Hepes (pH 8.0) 100mM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
Primer 7 or primer 5 200nM
Primer
8 or primer 6 200nM
SYBR Green I 0.1X
NaPPi 225μM
Mg(OAc) 2 2.5mM
Ung 2U
Tth stores damping fluid (0.2%Tween) 6％v/v
GDSE CS5DNA polysaccharase 5nM
Linearizing wild plasmid DNA 10 6Copy
Notice that " GDSE CS5 archaeal dna polymerase " refers to G46E D640G S671F E678G CS5DNA polysaccharase.In addition, each reaction volume is added to 50 μ l with the DEPC treated water.
The reacted constituent that changes comprises non-sealing primer (reaction of " nonocclusive " expression among Figure 21) and with 2 '-primer that phosphoric acid-C or 2 '-phosphoric acid-A seals (promptly comprise 2 of phosphate group '-terminating nucleotide) in 2 ' position.In addition, 10 6, 10 5, 10 4, 10 3, 10 2, 10 1Or the linearizing mutant K-Ras plasmid DNA of 0 copy (NTC reaction) (being respectively 10e6c, 10e5c, 10e4c, 10e3c, 10e2c, 10elc and NTC among Figure 21) also is added in the reaction.The correlator sequence of mutant plasmid DNA is mated fully with sealing primer and non-sealing primer both.Further, dilution in the 1 μ l HIV diluents (seeing above) of mutant K-Ras plasmid DNA in 1 μ l HIV diluents (seeing above) or " NTC " reaction.In addition, 10 6The linearizing wild-type K-Ras plasmid DNA of copy is present in institute and responds.Wild-type K-Ras plasmid DNA and mutant plasmid DNA are except that coincideing with sequence 3 ' base (dC) generation C:C mispairing of least significant end in the primer 5 and 7.Sealing and non-sealing primer are to all producing the amplicon of 92bp on mutant linearization plasmid template.
Embodiment XI: use the K-RAS plasmid template amplification of the various enzymes of varied concentration
K-Ras plasmid template with the various enzymes of varied concentration increases.Figure 22 shows for various enzymes that use in these reactions and the observed threshold circulation of concentration (x axle) (C T) value (y axle) chart.These data presentation, for example, the PAP amplification efficiency that uses certain enzyme described herein to obtain to improve.
Reaction is to use ABI 5700 sequence detection systems to carry out with following temperature distribution:
50 ℃ 2 minutes
93 ℃ 1 minute
92 ℃, 15 seconds → 60 ℃, 2 minutes * 60 circulations
Following reaction conditions is total by being responded:
Mother liquor composition Concentration
Hepes (pH 8.0) 100mM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
Primer
9 200nM
Primer
10 200nM
SYBR Green I 0.1X
NaPPi 225μM
Mg(OAc) 2 2.5mM
Ung 2U
Tth stores damping fluid (0.2%Tween) 6％v/v
Linearizing K-Ras plasmid DNA 10 4Copy
Reacted constituent comprises the primer (promptly comprising 2 ' of phosphate group-terminating nucleotide in 2 ' position) with 2 '-phosphoric acid-U or 2 '-phosphoric acid-A sealing.Primer is to producing the amplicon of 92bp on linearizing K-Ras plasmid template.In addition, each reaction volume is added to 50 μ l with diethylpyrocarbonate (DEPC) treated water.
By following to every kind of independent polysaccharase optimization polymerase concentration and KOAc concentration
Polysaccharase Polymerase concentration (nM) KOAc(mM)
GLQDSE 5,10,15,20,30 or 40nM 110
GLDSE 5,10,15,20,30 or 40nM 25
GLE 5,10,15,20,30 or 40nM 25
Note, " GLQDSE " refers to G46E L329A Q601R D640G S671F E678G CS5DNA polysaccharase, " GLDSE " refers to G46E L329A D640G S671F E678G CS5DNA polysaccharase, and " GLE " refers to G46E E678G CS5 archaeal dna polymerase.
Embodiment XII: hepatitis C virus (HCV) RNA of more non-sealing and sealing RT primer Reverse transcription (RT) to cDNA
The relatively extension and the extension of sealing primer of non-sealing HCV RT primer on the HCV RNA template in reverse transcription reaction.These RT relatively are to use various polysaccharases to carry out.Be explanation, Figure 23 be show for these uses comprise 5 '-PCR in real time of nuclease probe measures the chart of observed threshold circulation (Ct) values of the various enzymes (x axle) that use in the reaction of cDNA (y axle).
Following reaction conditions is total for all RT reactions:
The RT mother liquor composition Concentration
Hepes pH 8.0 100mM
KOAc 100mM
DMSO
4％(v/v)
Primer 1 or 2 200nM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
UNG 0.2 unit
Mn(OAc) 2 1mM
PPi 175uM
The reacted constituent that changes comprises the non-sealing primer of the 3 '-OH reaction of expression (among the Figure 23: 3 ' OH primer (nonocclusive) ") and with 2 '-primer that phosphoric acid-A or 2 '-single phosphoric acid-3 '-the hydroxyadenosine acid seal is closed (promptly comprise in 2 ' position 2 of phosphate group '-terminating nucleotide, the reaction of representing referring to " 2 ' PO4 (sealing) " among Figure 23).Further, more following polysaccharase condition (referring to Figure 23) in the cDNA reaction:
Z05 archaeal dna polymerase (13nM)
GLQDSE CS5 archaeal dna polymerase (100nM) and GLQDS CS5 archaeal dna polymerase (25nM)
GLQDSE CS5 archaeal dna polymerase (50nM) and GLQDS CS5 archaeal dna polymerase (50nM
Wherein " GLQDSE CS5 archaeal dna polymerase " refers to G46E L329A Q601RD640G S671F E678G CS5 archaeal dna polymerase, and " GLQDS CS5 archaeal dna polymerase " refers to G46E L329A Q601R D640G S671F CS5 archaeal dna polymerase.In addition, each reaction volume is added to 20 μ l with diethylpyrocarbonate (DEPC) treated water.
RT is reflected in ABI 9600 thermal cyclers and hatched 60 minutes in 60 ℃.After RT was hatched, RT was reflected in the DEPC treated water and dilutes 100 times.The existence that comes the real-time HCV PCR based on 5 ' nuclease probe of the HCV cDNA product in the particular measurement RT reaction to react to confirm cDNA with design and quantitatively.These reactions use ABI Prism 7700 sequential detectors to carry out with following temperature distribution:
50 ℃ 2 minutes
95 ℃ 15 seconds → 60 ℃ 1 minute * 50 circulation.
Embodiment XIII: be used for the two-way PAP that the BRAF sudden change detects
Figure 24 shows the PCR growth curve of the BRAF oncogene amplification that produces when carrying out two-way PAP.The accumulation fluorescence of x axle display standardization and y axle show the circulation of PAP pcr amplification.More particularly, these data are using 3 '-terminal nucleotide and the accurate site of sudden change eclipsed 2 '-primer of terminator sealing produces when determining the sudden change specific amplification of T → A sudden change that the V599E codon in the BRAF oncogene changes (referring to, Brose etc. (2002) Cancer Res 62:6997-7000).When the special primer of wild-type sequence and wildtype target or mutant target react, have only wildtype target to be detected.On the contrary, when the sequence-specific primer of mutant and wildtype target or mutant target react, have only the mutant target to be detected.
Following reaction conditions is total for all RT reactions:
Composition Concentration
Hepes pH 8.0 100mM
KOAc 100mM
Glycerine 3.5％v/v
Primers F 5W or F5M 200nM
Primer R5W or R5M 200nM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
UNG
1 unit
PPi 175uM
GLQDSE 15nM
SYBR I/ carboxyl rhodamine 1/100,000(0.1×)
Mg(OAc) 2 3.0mM
Wherein " GLQDSE " refers to G46E L329A Q601R D640G S671F E678G CS5DNA polysaccharase.
The reacted constituent that changes comprises with 2 '-phosphoric acid-A, 2 '-single phosphoric acid-3 '-hydroxyadenosine acid, 2 '-wild-type BRAF primer that phosphoric acid-U or 2 '-single phosphoric acid-3 '-hydroxyuridine acid seal is closed (promptly comprise in 2 ' position 2 of phosphate group '-terminating nucleotide, usefulness " F5W/R5W " mark in Figure 24).
In addition, each reaction volume is added to 50 μ l with the DEPC treated water.Wild-type reaction (in Figure 24 with " WT " mark) comprises the linearizing DNA plasmid of BRAF wild-type sequence and mutant reaction (in Figure 24 with " MT " mark) comprises the linearizing DNA plasmid of BRAF mutant sequence.Negative reaction (in Figure 24 with " NEG " mark) comprises do not have the HIV of DNA diluents (10mM Tris, 0.1mM EDTA, 20 μ g/mL Poly A, 0.09%NaN 3).Combination of primers among the PCR produces the amplicon of 50bp.Further, reaction uses ABI Prism7700 sequential detector carrying out with following temperature distribution:
50 ℃ 1 minute
93 ℃ 1 minute
90 ℃ 15 seconds
60 ℃ of 150 seconds → * 60 circulation.
Embodiment XIV: the detection of fluorescence PAP releasing product
This embodiment portentous has illustrated to relate to and sealed the real-time monitoring scheme of PAP activatory that primer causes that detectable signal produces when primer is activated and extend.
The structure of 3 ' terminated double-tagging Oligonucleolide primers:
Following primer QX comprises to append to its quencher dye molecule Black Hole from 3 ' terminal the 13rd Nucleotide (A)
Figure A20078003877200651
(BHQ) (Biosearch Technologies, DNA oligonucleotide Inc.).
The QX Oligonucleolide primers mixes with complementary oligonucleotide R1 (referring to as follows) in solution, and they form the heteroduplex body like this.The reagent mix of archaeal dna polymerase that comprises fluorescein-labeled ribodesose VITAMIN B4 four phosphoric acid (be fluorescein-labeled 2 '-terminating nucleotide) especially and can mix four phosphoric acid of above-mentioned mark that this duplex further provides with following table 10.Application No. 10/879 referring to " the SYNTHESIS AND COMPOSITIONS OF2 '-TERMINATOR NUCLEOTIDES " by name that submitted on June 28th, 2004, the Application No. 10/879,493 of " the 2 '-TERMINATOR NUCLEOTIDE-RELATEDMETHODS AND SYSTEMS " by name that submitted on June 28th, 494 and 2004.Under 60 ℃ of temperature, hatch this mixture, for example, can make that 3 of sequence QX ' is terminal to extend Nucleotide in template guided mode in one hour, as a result at least a portion QX oligonucleotide its 3 ' end extended fluorescein-labeled ribodesose VITAMIN B4-2 '-phosphoric acid nucleoside acid, below with primer QX FAMExpression.
Table 10
Mother liquor composition Concentration
Hepes pH 8.3 50mM
KOAc 100mM
Glycerine
8％(w/v)
Primer QX 10μM
Oligonucleotide R1 15μM
Fluorescein dA4P 15μM
G46E L329A E678G CS5 archaeal dna polymerase 50nM
Mg(OAc) 2 2.5mM
The new primer QX that prolongs FAMUse many purification process known to those skilled in the art purifying from said mixture.Above-mentioned can be from this mixture purifying primer QX FAMAn example of method be high performance liquid chromatography (HPLC).Select HPLC purifying parameter so that primer QX FAMGoods are substantially free of primer QX and fluorescein-labeled VITAMIN B4 four phosphoric acid that do not extend.Dual HPLC (anti-phase and anionresin HPLC) is a kind of method of the above-mentioned molecule of known purifying.
Behind the purifying, on same oligonucleotide, comprise BHQ quencher molecule and fluorescein molecule such as primer QX FAMMolecule demonstrate usually because the fluorescein signal of the inhibition that energy is caused by BHQ2 " quencher " molecular absorption.
At random, primer QX FAMAccording to, for example, the description chemosynthesis in the U.S. Patent Publication No. 2007/0219361.
The sequence that relates in the present embodiment is as follows:
Primer QX 5 '-GCAAGCACCCTATCA QGGCAGTACCACA-3 ' (SEQ IDNO:73)
(wherein Q represents the existence of BHQ molecule)
R1 3’-PCGTTCGTGGGATAGTCCGTCATGGTGTT-5’(SEQ ID NO：74)
(wherein P represents 3 ' phosphoric acid)
Primer QX FAM5 '-GCAAGCACCCTATCA QGGCAGTACCACA F-3 ' (SEQ IDNO:75)
(wherein Q represents the existence of BHQ molecule, and F represents fluorescein-labeled 2 ' Vitamin B4)
Primer HC2 5 '-GCAGAAAGCGTCTAGCCATGGCTTA-3 ' (SEQ ID NO:76).
The use of primer among the PCR.
With above-described primer QX FAMCombine with the reagent in the table 11.
Table 11
Composition Concentration
Hepes pH 8.0 100mM
KOAc 100mM
Glycerine 3.5％(v/v)
DMSO 5％(v/v)
Primer QX FAM 150nM
Primer HC2 150nM
dATP 200μM
dCTP 200μM
dGTP 200μM
dTTP 30μM
dUTP 300μM
UNG
1 unit
PPi 175μM
GLQDSE 15nM
Target sequence
10 6Copy
Mg(OAc) 2 3.0mM
In addition, each reaction volume is added to 50 μ l with the DEPC treated water.Some reactions comprise the target sequence as the pcr amplification substrate, and other do not comprise target.For example, target can be and the identical dna sequence dna in HCV genome 5 ' UTR zone.The combination of these primers estimates to produce the amplicon of about 244bp among the PCR.
Reaction can use ABI Prism 7700 sequential detectors to carry out with following temperature distribution:
50 ℃ 1 minute
93 ℃ 1 minute
90 ℃ 15 seconds
60 ℃ of 150 " → * 60 circulation
For carrying out the improvement of above-mentioned PCR, fluorescein terminated primer QX FAMPAP activation be necessary, and will cause the excision of fluorescein-labeled deoxyadenine four phosphoric acid moleculeses.Above-mentioned release is estimated in the approximately increase of 520nm wavelength generation fluorescent signal.When PCR carries out, monitor the signal of about 520nm wavelength, expectation is observed the increase of fluorescence and in the reaction that does not comprise target, do not observe fluorescence and increase in those comprise the reaction of target nucleic acid.
Embodiment XV:D580K, D580L, D580R and D580T sudden change are to the Z05 archaeal dna polymerase Extend the effect of speed
The effect of speed is extended in the various replacements of having measured the D580 site to the Z05 archaeal dna polymerase.At first,, utilize overlapping pcr on the Z05 archaeal dna polymerase, to produce sudden change according to previously described, and purifying and quantitative mutant enzyme.According to the description in the foregoing description II and other place, use Mg + 2And Mn + 2The both is as the metal cofactor, is determined at extension speed on M13 (single stranded DNA) template that has caused by the increase that detects SYBR Green I fluorescence.In the present embodiment, reaction mixture comprises 50mM Hepes pH 8.3,40mMKOAc, 1mM Mn (OAc) 2Or 2.5mM Mg (OAc) 2, 1.25%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM Tris pH 8.0,0.1mM EDTA, 1mM DTT, 0.5%Tween 20), 1%DMSO, 0.6 * SYBR Green I, the M13 that 1.0nM has caused and or 5nM enzyme.Nucleotide adds with the final concentration of 0.2mM dGTP, 0.2mM dTTP, 0.2mMdCTP and 0.2mM dATP.The parallel reaction that does not comprise Nucleotide also is established.Total overall reaction runs on the 384 hole thermal cycler flat boards in quadruplicate with 20 μ l volumes.Fluoroscopic examination is used in extending in the dynamic thermal cycler in 64 ℃ of the M13 template that has caused down, obtains reading in per 15 seconds.Calculate same reaction repeated experiments mean number and deduct parallel negative Nucleotide reaction.From the data that produce, extend speed with the linear regression analysis estimation.The results are shown in following table 12:
Table 12
The extension speed of Z05 D580X mutant.
(based on the change of fluorescence, arbitrary unit)
Figure A20078003877200691
All 5 seed amino acids replacements in the site 580 of data presentation Z05 archaeal dna polymerase all cause under the condition of test extends speed faster.
The use of various mutant Z05 archaeal dna polymerases among the embodiment XVI:RT-PCR
Based on Mn 2+RT: Mn is being arranged + 2Situation under assessment sudden change D580G, D580K and D580R for the effect of RT-PCR efficient.Reaction all comprises following component: 55mM Hepes pH 8.3,4%v/v glycerine, 5%v/v DMSO, 110mM KOAc, 2.7mMMn (OAc) 2, 3.6%v/v storage damping fluid (50%v/v glycerine, 100mM KCl, 20mM TrispH 8.0,0.1mM EDTA, 1mM DTT, 0.2%Tween 20), 0.04 unit/μ l UNG, dATP, dCTP, each 0.45mM of dUTP, dGTP; Every kind of primer 750nM, wherein every kind of primer comprises tert.-butylbenzene methyl dA at 3 '-end; With the TaqMan probe of 150nM with cyclohexyl-FAM, blackhole quencher (BHQ-2), 3 '-phosphoric acid mark.Two kinds of primers produce the amplicon of 241bp jointly on the HCV-1B transcript.Per 100 μ l reaction adds 10 5Copy rna transcription thing HCV-1B.
There is not the parallel reaction of transcript to be established yet.Adding every kind of enzyme is 27nM to final concentration.Be reflected in the dynamic thermal cycler of Roche LC480 and move.Thermal cycle conditions is: 50 ℃ 5 minutes (" UNG " step); 66 ℃ 2,5 or 30 minutes (" RT " step); 95 ℃ of 2 round-robin 15 seconds and subsequently 58 ℃ 50 seconds; Then 91 ℃ of 50 round-robin 15 seconds and subsequently 58 ℃ 50 seconds.
Table 13 shows the Ct value that has increased access to from the FAM signal owing to the cutting of TaqMan probe:
Table 13
Figure A20078003877200701
The result shows that these three kinds of sudden changes in D580 site allow that the shorter RT time keeps suitable RT efficient simultaneously.
Based on Mg 2+RT: relatively suddenly change D580G and D580K and ES112 (Z05 E683R) are having Mg + 2Situation under carry out the ability of RT-PCR.Known to ES112, parent enzyme and Z05 archaeal dna polymerase carry out based on Mg with the Ct value of remarkable delay + 2RT-PCR, in this research again the test.The condition of using is consistent with above-mentioned firm description, except KOAc changes 50mM into, and Mn (OAc) 2Replace with 2mM Mg (OAC) 2, and enzyme concn is kept to 10nM.Thermal cycle conditions is identical, except only having tested 30 minutes RT time.
Table 14 shows the Ct value that has increased access to from the FAM signal owing to the cutting of TaqMan probe:
Table 14
Figure A20078003877200702
The result shows that the D580G mutant is to carry out based on Mg with the roughly the same efficient of ES112 + 2RT PCR, and the D580K mutant causes the Ct value that significantly shifts to an earlier date, demonstrates the RT efficient that improves greatly under these conditions.
Should be appreciated that embodiment described herein and embodiment just are used for the illustrative purpose, and its various modifications or change to be inspired and give those skilled in the art, and be included in the scope of the application's spirit and scope and additional claim.
Sequence table
<110>F.Hoffmann-La Roche AG
Roche Diagnostics GmbH
＜120〉mutant DNA polymerases and methods involving
<130>23146 WO-KOE
＜140〉do not specify yet
＜141〉do not specify yet
<150>US 60/852,882
<151>2006-10-18
<160>76
<170>PatentIn version 3.3
<210>1
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif a
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Gln, Thr, Met, any amino acid outside Gly or the Leu
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Glu or Asp
<400>1
Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa Thr Tyr Xaa Xaa
1 5 10 15
<210>2
<211>13
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif b
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Asp, any amino acid outside Glu or the Asn
<400>2
Thr Gly Arg Leu Ser Ser Xaa Xaa Pro Asn Leu Gln Asn
1 5 10
<210>3
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉any amino acid outside Xaa=Ile or the Leu
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Ser, Ala, any amino acid outside Val or the Gly
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>3
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>4
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the Polymerase Structure territory of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of thermus thermophilus (Tth)
The avtive spot zone
<400>4
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>5
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of Thermus caldophilus (Tca)
The avtive spot zone of structural domain
<400>5
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Asn Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>6
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉Polymerase Structure of the archaeal dna polymerase that relies on from thermally-stabilised family Aform DNA from the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone in territory
<400>6
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>7
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉work in the Polymerase Structure territory of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of thermus aquaticus (Taq)
The property site areas
<400>7
Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>8
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉from the dwell activity in Polymerase Structure territory of the archaeal dna polymerase that the thermally-stabilised family Aform DNA of hot bacterium (Tf1) relies on of Huang
Site areas
<400>8
Ile Val Asp Arg Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Ile Asp Pro Leu Pro Ala Leu Val His Pro Lys Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Glu Gly Trp Val Leu Val
65 70 75 80
Val Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>9
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉work in the Polymerase Structure territory of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the thread hot bacterium (Tfi) that dwells
The property site areas
<400>9
Ile Val Gly Arg Ile Leu Glu Tyr Arg Glu Leu Met Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Pro Leu Pro Arg Leu Val His Pro Lys Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Lys Ala Phe Ile Ala Glu Glu Gly His Leu Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>10
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉Polymerase Structure of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification sps17 (sps17) of dwelling
The avtive spot zone in territory
<400>10
Ile Val Gly Arg Ile Leu Glu Tyr Arg Glu Leu Met Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Pro Leu Pro Arg Leu Val His Pro Lys Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Lys Ala Phe Ile Ala Glu Glu Gly His Leu Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>11
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉work in the Polymerase Structure territory of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of Thermotoga maritima (Tma)
The property site areas
<400>11
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>12
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉from the dwell Polymerase Structure of the archaeal dna polymerase that the thermally-stabilised family Aform DNA of thermobacillus (Tne) relies on of Naples
The avtive spot zone in territory
<400>12
Ile Val Pro Leu Ile Leu Glu Phe Arg Lys Ile Leu Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Thr Leu Pro Lys Leu Val Asn Pro Lys Thr Gly Arg
20 25 30
Phe His Ala Ser Phe His Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asp Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>13
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉from the dwell Polymerase Structure territory of archaeal dna polymerase that the thermally-stabilised family Aform DNA of hot chamber bacterium (Taf) relies on, Africa
The avtive spot zone
<400>13
Ile Ala Lys Leu Leu Leu Glu Tyr Arg Lys Tyr Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ser Ile Pro Leu Ser Ile Asn Arg Lys Thr Asn Arg
20 25 30
Val His Thr Thr Phe His Gln Thr Gly Thr Ser Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asn Pro Asn Leu Gln Asn Leu Pro Thr Arg Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Val Arg Pro Gln Arg Gln Asp Trp Trp Ile
65 70 75 80
Leu Gly Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>14
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉come the Polymerase Structure territory of archaeal dna polymerase that the thermally-stabilised family Aform DNA of the hard genus bacillus of self-heating (Bca) relies on
The avtive spot zone
<400>14
Val Glu Asn Ile Leu Gln His Tyr Arg Gln Leu Gly Lys Leu Gln Ser
1 5 10 15
Thr Tyr Ile Glu Gly Leu Leu Lys Val Val Arg Pro Asp Thr Lys Lys
20 25 30
Val His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Ser
35 40 45
Ser Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gly
50 55 60
Arg Lys Ile Arg Gln Ala Phe Val Pro Ser Glu Ser Asp Trp Leu Ile
65 70 75 80
Phe Ala Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>15
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉the avtive spot zone in the Polymerase Structure territory of the archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA
<400>15
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>16
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉the avtive spot zone in the Polymerase Structure territory of the archaeal dna polymerase CS6 that relies on from chimeric heat-stable DNA
<400>16
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>17
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉avtive spot zone, the Polymerase Structure territory concensus sequence (Cons) of the archaeal dna polymerase of heat-stable DNA dependence
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Val, Ile, Ala or Glu
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Glu, Pro, Gly, Asp, Lys or Asn
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Leu, Lys, Arg or Ile
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(10)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Gln, Thr, Met, Gly or Leu
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(19)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(20)
＜223〉Xaa=Glu or Asp
<220>
<221>MOD_RES
<222>(21)
＜223〉Xaa=Pro, Ala, Thr, Ser or Gly
<220>
<221>MOD_RES
<222>(22)
＜223〉Xaa=Leu or Ile
<220>
<221>MOD_RES
<222>(24)
＜223〉Xaa=Lys, Arg, Ser, Leu, Ala or Asp
<220>
<221>MOD_RES
<222>(25)
＜223〉Xaa=Leu, Met, Val or Ser
<220>
<221>MOD_RES
<222>(26)
＜223〉Xaa=Val or Ile
<220>
<221>MOD_RES
<222>(27)
＜223〉Xaa=Hi s, Asn or Arg
<220>
<221>MOD_RES
<222>(28)
＜223〉Xaa=Pro or Arg
<220>
<221>MOD_RES
<222>(29)
＜223〉Xaa=Lys, Arg, Asn or Asp
<220>
<221>MOD_RES
<222>(31)
＜223〉Xaa=Gly, Lys or Asn
<220>
<221>MOD_RES
<222>(32)
＜223〉Xaa=Arg or Lys
<220>
<221>MOD_RES
<222>(33)
＜223〉Xaa=Leu, Ile, Val or Phe
<220>
<221>MOD_RES
<222>(35)
＜223〉Xaa=Thr or Ala
<220>
<221>MOD_RES
<222>(36)
＜223〉Xaa=Arg, Ser, Thr or Ile
<220>
<221>MOD_RES
<222>(38)
＜223〉Xaa=Asn or His
<220>
<221>MOD_RES
<222>(40)
＜223〉Xaa=Thr or Ala
<220>
<221>MOD_RES
<222>(41)
＜223〉Xaa=Ala, Gly or Leu
<220>
<221>MOD_RES
<222>(43)
＜223〉Xaa=Ala, Ser or Gln
<220>
<221>MOD_RES
<222>(50)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(51)
＜223〉Xaa=Asp, Glu or Asn
<220>
<221>MOD_RES
<222>(57)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(59)
＜223〉Xaa=Val, Thr or Ile
<220>
<221>MOD_RES
<222>(60)
＜223〉Xaa=Arg or Lys
<220>
<221>MOD_RES
<222>(61)
＜223〉Xaa=Thr, Ser or Leu
<220>
<221>MOD_RES
<222>(62)
＜223〉Xaa=Pro or Glu
<220>
<221>MOD_RES
<222>(63)
＜223〉Xaa=Leu or Glu
<220>
<221>MOD_RES
<222>(65)
＜223〉Xaa=Gln, Lys or Arg
<220>
<221>MOD_RES
<222>(66)
＜223〉Xaa=Arg, Glu or Lys
<220>
<221>MOD_RES
<222>(69)
＜223〉Xaa=Lys, Arg or Gln
<220>
<221>MOD_RES
<222>(71)
＜223〉Xaa=Phe, Ile or Val
<220>
<221>MOD_RES
<222>(72)
＜223〉Xaa=Val, Ile or Arg
<220>
<221>MOD_RES
<222>(73)
＜223〉Xaa=Ala or Pro
<220>
<221>MOD_RES
<222>(74)
＜223〉Xaa=Gln or non-existent
<220>
<221>MOD_RES
<222>(75)
＜223〉Xaa=Glu, Asp or Arg
<220>
<221>MOD_RES
<222>(76)
＜223〉Xaa=Pro, Glu, Ala, Ser or Asp
<220>
<221>MOD_RES
<222>(77)
＜223〉Xaa=Gly, Asp or Asn
<220>
<221>MOD_RES
<222>(78)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(79)
＜223〉Xaa=Trp, Leu, Ala or Val
<220>
<221>MOD_RES
<222>(80)
＜223〉Xaa=Leu or Ile
<220>
<221>MOD_RES
<222>(81)
＜223〉Xaa=Val, Leu or Phe
<220>
<221>MOD_RES
<222>(82)
＜223〉Xaa=Ala, Ser, Gly or Val
<220>
<221>MOD_RES
<222>(83)
＜223〉Xaa=Leu or Ala
<220>
<221>MOD_RES
<222>(92)
＜223〉Xaa=Val or Ile
<400>17
Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa
1 5 10 15
Thr Tyr Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa
20 25 30
Xaa His Xaa Xaa Phe Xaa Gln Xaa Xaa Thr Xaa Thr Gly Arg Leu Ser
35 40 45
Ser Xaa Xaa Pro Asn Leu Gln Asn Xaa Pro Xaa Xaa Xaa Xaa Xaa Gly
50 55 60
Xaa Xaa Ile Arg Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg Xaa
85 90
<210>18
<211>893
<212>PRT
＜213〉artificial
<220>
＜223〉the archaeal dna polymerase CS5 of chimeric heat-stable DNA dependence
<400>18
Met Lys Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe
50 55 60
Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu
65 70 75 80
Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln
85 90 95
Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu
100 105 110
Glu Val Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys
115 120 125
Lys Ala Glu Arg Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg
130 135 140
Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu
145 150 155 160
Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys
165 170 175
Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp
180 185 190
Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu
195 200 205
Leu Lys Glu Trp Gly Ser Leu Glu Asn Ile Leu Lys Asn Leu Asp Arg
210 215 220
Val Lys Pro Glu Ser Val Arg Glu Arg Ile Lys Ala His Leu Glu Asp
225 230 235 240
Leu Lys Leu Ser Leu Glu Leu Ser Arg Val Arg Ser Asp Leu Pro Leu
245 250 255
Glu Val Asp Phe Ala Arg Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg
260 265 270
Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285
Leu Leu Glu Glu Ser Glu Pro Val Gly Tyr Arg Ile Val Lys Asp Leu
290 295 300
Val Glu Phe Glu Lys Leu Ile Glu Lys Leu Arg Glu Ser Pro Ser Phe
305 310 315 320
Ala Ile Asp Leu Glu Thr Ser Ser Leu Asp Pro Phe Asp Cys Asp Ile
325 330 335
Val Gly Ile Ser Val Ser Phe Lys Pro Lys Glu Ala Tyr Tyr Ile Pro
340 345 350
Leu His His Arg Asn Ala Gln Asn Leu Asp Glu Lys Glu Val Leu Lys
355 360 365
Lys Leu Lys Glu Ile Leu Glu Asp Pro Gly Ala Lys Ile Val Gly Gln
370 375 380
Asn Leu Lys Phe Asp Tyr Lys Val Leu Met Val Lys Gly Val Glu Pro
385 390 395 400
Val Pro Pro Tyr Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro
405 410 415
Asn Glu Lys Lys Phe Asn Leu Asp Asp Leu Ala Leu Lys Phe Leu Gly
420 425 430
Tyr Lys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Phe Pro Leu
435 440 445
Phe Gly Phe Ser Phe Ala Asp Val Pro Val Glu Lys Ala Ala Asn Tyr
450 455 460
Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Thr Leu Ser
465 470 475 480
Leu Lys Leu His Glu Ala Asp Leu Glu Asn Val Phe Tyr Lys Ile Glu
485 490 495
Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu Asn Gly Val Tyr
500 505 510
Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu Glu Tyr Gly Lys Lys
515 520 525
Leu Glu Glu Leu Ala Glu Glu Ile Tyr Arg Ile Ala Gly Glu Pro Phe
530 535 540
Asn Ile Asn Ser Pro Lys Gln Val Ser Arg Ile Leu Phe Glu Lys Leu
545 550 555 560
Gly Ile Lys Pro Arg Gly Lys Thr Thr Lys Thr Gly Asp Tyr Ser Thr
565 570 575
Arg Ile Glu Val Leu Glu Glu Leu Ala Gly Glu His Glu Ile Ile Pro
580 585 590
Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser Thr Tyr Ile
595 600 605
Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg Ile His Ala
610 615 620
Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp
625 630 635 640
Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile
645 650 655
Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile Val Ser Ala
660 665 670
Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp
675 680 685
Glu Asn Leu Leu Arg Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu
690 695 700
Thr Ala Ser Arg Ile Phe Asn Val Lys Pro Glu Glu Val Thr Glu Glu
705 710 715 720
Met Arg Arg Ala Gly Lys Met Val Asn Phe Ser Ile Ile Tyr Gly Val
725 730 735
Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Val Pro Val Lys Glu Ala
740 745 750
Glu Lys Met Ile Val Asn Tyr Phe Val Leu Tyr Pro Lys Val Arg Asp
755 760 765
Tyr Ile Gln Arg Val Val Ser Glu Ala Lys Glu Lys Gly Tyr Val Arg
770 775 780
Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro Gln Leu Met Ala Arg Asp
785 790 795 800
Arg Asn Thr Gln Ala Glu Gly Glu Arg Ile Ala Ile Asn Thr Pro Ile
805 810 815
Gln Gly Thr Ala Ala Asp Ile Ile Lys Leu Ala Met Ile Glu Ile Asp
820 825 830
Arg Glu Leu Lys Glu Arg Lys Met Arg Ser Lys Met Ile Ile Gln Val
835 840 845
His Asp Glu Leu Val Phe Glu Val Pro Asn Glu Glu Lys Asp Ala Leu
850 855 860
Val Glu Leu Val Lys Asp Arg Met Thr Asn Val Val Lys Leu Ser Val
865 870 875 880
Pro Leu Glu Val Asp Val Thr Ile Gly Lys Thr Trp Ser
885 890
<210>19
<211>893
<212>PRT
＜213〉artificial
<220>
＜223〉the archaeal dna polymerase CS6 of chimeric heat-stable DNA dependence
<400>19
Met Lys Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe
50 55 60
Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu
65 70 75 80
Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln
85 90 95
Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu
100 105 110
Glu Val Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys
115 120 125
Lys Ala Glu Arg Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg
130 135 140
Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu
145 150 155 160
Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys
165 170 175
Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp
180 85 190
Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu
195 200 205
Leu Lys Glu Trp Gly Ser Leu Glu Asn Ile Leu Lys Asn Leu Asp Arg
210 215 220
Val Lys Pro Glu Ser Val Arg Glu Arg Ile Lys Ala His Leu Glu Asp
225 230 235 240
Leu Lys Leu Ser Leu Glu Leu Ser Arg Val Arg Ser Asp Leu Pro Leu
245 250 255
Glu Val Asp Phe Ala Arg Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg
260 265 270
Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285
Leu Leu Glu Glu Ser Glu Pro Val Gly Tyr Arg Ile Val Lys Asp Leu
290 295 300
Val Glu Phe Glu Lys Leu Ile Glu Lys Leu Arg Glu Ser Pro Ser Phe
305 310 315 320
Ala Ile Ala Leu Ala Thr Ser Ser Leu Asp Pro Phe Asp Cys Asp Ile
325 330 335
Val Gly Ile Ser Val Ser Phe Lys Pro Lys Glu Ala Tyr Tyr Ile Pro
340 345 350
Leu His His Arg Asn Ala Gln Asn Leu Asp Glu Lys Glu Val Leu Lys
355 360 365
Lys Leu Lys Glu Ile Leu Glu Asp Pro Gly Ala Lys Ile Val Gly Gln
370 375 380
Asn Leu Lys Phe Asp Tyr Lys Val Leu Met Val Lys Gly Val Glu Pro
385 390 395 400
Val Pro Pro Tyr Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro
405 410 415
Asn Glu Lys Lys Phe Asn Leu Asp Asp Leu Ala Leu Lys Phe Leu Gly
420 425 430
Tyr Lys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Phe Pro Leu
435 440 445
Phe Gly Phe Ser Phe Ala Asp Val Pro Val Glu Lys Ala Ala Asn Tyr
450 455 460
Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Thr Leu Ser
465 470 475 480
Leu Lys Leu His Glu Ala Asp Leu Glu Asn Val Phe Tyr Lys Ile Glu
485 490 495
Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu Asn Gly Val Tyr
500 505 510
Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu Glu Tyr Gly Lys Lys
515 520 525
Leu Glu Glu Leu Ala Glu Glu Ile Tyr Arg Ile Ala Gly Glu Pro Phe
530 535 540
Asn Ile Asn Ser Pro Lys Gln Val Ser Arg Ile Leu Phe Glu Lys Leu
545 550 555 560
Gly Ile Lys Pro Arg Gly Lys Thr Thr Lys Thr Gly Asp Tyr Ser Thr
565 570 575
Arg Ile Glu Val Leu Glu Glu Leu Ala Gly Glu His Glu Ile Ile Pro
580 585 590
Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser Thr Tyr Ile
595 600 605
Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg Ile His Ala
610 615 620
Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp
625 630 635 640
Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile
645 650 655
Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile Val Ser Ala
660 665 670
Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp
675 680 685
Glu Asn Leu Leu Arg Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu
690 695 700
Thr Ala Ser Arg Ile Phe Asn Val Lys Pro Glu Glu Val Thr Glu Glu
705 710 715 720
Met Arg Arg Ala Gly Lys Met Val Asn Phe Ser Ile Ile Tyr Gly Val
725 730 735
Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Val Pro Val Lys Glu Ala
740 745 750
Glu Lys Met Ile Val Asn Tyr Phe Val Leu Tyr Pro Lys Val Arg Asp
755 760 765
Tyr Ile Gln Arg Val Val Ser Glu Ala Lys Glu Lys Gly Tyr Val Arg
770 775 780
Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro Gln Leu Met Ala Arg Asp
785 790 795 800
Arg Asn Thr Gln Ala Glu Gly Glu Arg Ile Ala Ile Asn Thr Pro Ile
805 810 815
Gln Gly Thr Ala Ala Asp Ile Ile Lys Leu Ala Met Ile Glu Ile Asp
820 825 830
Arg Glu Leu Lys Glu Arg Lys Met Arg Ser Lys Met Ile Ile Gln Val
835 840 845
His Asp Glu Leu Val Phe Glu Val Pro Asn Glu Glu Lys Asp Ala Leu
850 855 860
Val Glu Leu Val Lys Asp Arg Met Thr Asn Val Val Lys Leu Ser Val
865 870 875 880
Pro Leu Glu Val Asp Val Thr Ile Gly Lys Thr Trp Ser
885 890
<210>20
<211>2682
<212>DNA
＜213〉artificial
<220>
＜223〉the archaeal dna polymerase CS5 of chimeric heat-stable DNA dependence
<400>20
atgaaagcta tgttaccatt attcgaaccc aaaggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt cttcgccctg aagggcctca ccacgagccg gggcgaaccg 120
gtgcaggcgg tttacggctt cgccaagagc ctcctcaagg ccctgaagga ggacgggtac 180
aaggccgtct tcgtggtctt tgacgccaag gccccttcct tccgccacga ggcctacgag 240
gcctacaagg caggccgcgc cccgaccccc gaggacttcc cccggcagct cgccctcatc 300
aaggagctgg tggacctcct ggggtttact cgcctcgagg ttccgggctt tgaggcggac 360
gacgtcctcg ccaccctggc caagaaggcg gaaagggagg ggtacgaggt gcgcatcctc 420
accgccgacc gggaccttta ccagctcgtc tccgaccgcg tcgccgtcct ccaccccgag 480
ggccacctca tcaccccgga gtggctttgg gagaagtacg gccttaagcc ggagcagtgg 540
gtggacttcc gcgccctcgt gggggacccc tccgacaacc tccccggggt caagggcatc 600
ggggagaaga ccgccctcaa gctcctcaag gagtggggaa gcctggaaaa tatcctcaag 660
aacctggacc gggtgaagcc ggaaagcgtc cgggaaagga tcaaggccca cctggaagac 720
cttaagctct ccttggagct ttcccgggtg cgctcggacc tccccctgga ggtggacttc 780
gcccggaggc gggagcctga ccgggaaggg cttcgggcct ttttggagcg cttggagttc 840
ggcagcctcc tccacgagtt cggccttcta gaggagtccg aacccgttgg gtaccgtata 900
gttaaagacc tggttgaatt tgaaaaactc atagagaaac tgagagaatc tccttcgttc 960
gctatcgatt tggaaactag ttccctcgat cctttcgact gcgacattgt cggtatctct 1020
gtgtctttca aaccaaagga agcgtactac ataccactcc atcatagaaa cgcccagaac 1080
ctggacgaaa aagaggttct gaaaaagctc aaagaaattc tggaggaccc cggagcaaag 1140
atcgttggtc agaatttgaa attcgattac aaggtgttga tggtgaaggg tgttgaacct 1200
gttcctcctt acttcgacac gatgatagcg gcttaccttc ttgagccgaa cgaaaagaag 1260
ttcaatctgg acgatctcgc attgaaattt cttggataca aaatgacatc ttaccaagag 1320
ctcatgtcct tctcttttcc gctgtttggt ttcagttttg ccgatgttcc tgtagaaaaa 1380
gcagcgaact actcctgtga agatgcagac atcacctaca gactttacaa gaccctgagc 1440
ttaaaactcc acgaggcaga tctggaaaac gtgttctaca agatagaaat gccccttgtg 1500
aacgtgcttg cacggatgga actgaacggt gtgtatgtgg acacagagtt cctgaagaaa 1560
ctctcagaag agtacggaaa aaaactcgaa gaactggcag aggaaatata caggatagct 1620
ggagagccgt tcaacataaa ctcaccgaag caggtttcaa ggatcctttt tgaaaaactc 1680
ggcataaaac cacgtggtaa aacgacgaaa acgggagact attcaacacg catagaagtc 1740
ctcgaggaac ttgccggtga acacgaaatc attcctctga ttcttgaata cagaaagata 1800
cagaaattga aatcaaccta catagacgct cttcccaaga tggtcaaccc aaagaccgga 1860
aggattcatg cttctttcaa tcaaacgggg actgccactg gaagacttag cagcagcgat 1920
cccaatcttc agaacctccc gacgaaaagt gaagagggaa aagaaatcag gaaagcgata 1980
gttcctcagg atccaaactg gtggatcgtc agtgccgact actcccaaat agaactgagg 2040
atcctcgccc atctcagtgg tgatgagaat cttttgaggg cattcgaaga gggcatcgac 2100
gtccacactc taacagcttc cagaatattc aacgtgaaac ccgaagaagt aaccgaagaa 2160
atgcgccgcg ctggtaaaat ggttaatttt tccatcatat acggtgtaac accttacggt 2220
ctgtctgtga ggcttggagt acctgtgaaa gaagcagaaa agatgatcgt caactacttc 2280
gtcctctacc caaaggtgcg cgattacatt cagagggtcg tatcggaagc gaaagaaaaa 2340
ggctatgtta gaacgctgtt tggaagaaaa agagacatac cacagctcat ggcccgggac 2400
aggaacacac aggctgaagg agaacgaatt gccataaaca ctcccataca gggtacagca 2460
gcggatataa taaagctggc tatgatagaa atagacaggg aactgaaaga aagaaaaatg 2520
agatcgaaga tgatcataca ggtccacgac gaactggttt ttgaagtgcc caatgaggaa 2580
aaggacgcgc tcgtcgagct ggtgaaagac agaatgacga atgtggtaaa gctttcagtg 2640
ccgctcgaag tggatgtaac catcggcaaa acatggtcgt ga 2682
<210>21
<211>2682
<212>DNA
＜213〉artificial
<220>
＜223〉the archaeal dna polymerase CS6 of chimeric heat-stable DNA dependence
<400>21
atgaaagcta tgttaccatt attcgaaccc aaaggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt cttcgccctg aagggcctca ccacgagccg gggcgaaccg 120
gtgcaggcgg tttacggctt cgccaagagc ctcctcaagg ccctgaagga ggacgggtac 180
aaggccgtct tcgtggtctt tgacgccaag gccccttcct tccgccacga ggcctacgag 240
gcctacaagg caggccgcgc cccgaccccc gaggacttcc cccggcagct cgccctcatc 300
aaggagctgg tggacctcct ggggtttact cgcctcgagg ttccgggctt tgaggcggac 360
gacgtcctcg ccaccctggc caagaaggcg gaaagggagg ggtacgaggt gcgcatcctc 420
accgccgacc gggaccttta ccagctcgtc tccgaccgcg tcgccgtcct ccaccccgag 480
ggccacctca tcaccccgga gtggctttgg gagaagtacg gccttaagcc ggagcagtgg 540
gtggacttcc gcgccctcgt gggggacccc tccgacaacc tccccggggt caagggcatc 600
ggggagaaga ccgccctcaa gctcctcaag gagtggggaa gcctggaaaa tatcctcaag 660
aacctggacc gggtgaagcc ggaaagcgtc cgggaaagga tcaaggccca cctggaagac 720
cttaagctct ccttggagct ttcccgggtg cgctcggacc tccccctgga ggtggacttc 780
gcccggaggc gggagcctga ccgggaaggg cttcgggcct ttttggagcg cttggagttc 840
ggcagcctcc tccacgagtt cggccttcta gaggagtccg aacccgttgg gtaccgtata 900
gttaaagacc tggttgaatt tgaaaaactc atagagaaac tgagagaatc tccttcgttc 960
gcgatcgctc ttgcgactag ttccctcgat cctttcgact gcgacattgt cggtatctct 1020
gtgtctttca aaccaaagga agcgtactac ataccactcc atcatagaaa cgcccagaac 1080
ctggacgaaa aagaggttct gaaaaagctc aaagaaattc tggaggaccc cggagcaaag 1140
atcgttggtc agaatttgaa attcgattac aaggtgttga tggtgaaggg tgttgaacct 1200
gttcctcctt acttcgacac gatgatagcg gcttaccttc ttgagccgaa cgaaaagaag 1260
ttcaatctgg acgatctcgc attgaaattt cttggataca aaatgacatc ttaccaagag 1320
ctcatgtcct tctcttttcc gctgtttggt ttcagttttg ccgatgttcc tgtagaaaaa 1380
gcagcgaact actcctgtga agatgcagac atcacctaca gactttacaa gaccctgagc 1440
ttaaaactcc acgaggcaga tctggaaaac gtgttctaca agatagaaat gccccttgtg 1500
aacgtgcttg cacggatgga actgaacggt gtgtatgtgg acacagagtt cctgaagaaa 1560
ctctcagaag agtacggaaa aaaactcgaa gaactggcag aggaaatata caggatagct 1620
ggagagccgt tcaacataaa ctcaccgaag caggtttcaa ggatcctttt tgaaaaactc 1680
ggcataaaac cacgtggtaa aacgacgaaa acgggagact attcaacacg catagaagtc 1740
ctcgaggaac ttgccggtga acacgaaatc attcctctga ttcttgaata cagaaagata 1800
cagaaattga aatcaaccta catagacgct cttcccaaga tggtcaaccc aaagaccgga 1860
aggattcatg cttctttcaa tcaaacgggg actgccactg gaagacttag cagcagcgat 1920
cccaatcttc agaacctccc gacgaaaagt gaagagggaa aagaaatcag gaaagcgata 1980
gttcctcagg atccaaactg gtggatcgtc agtgccgact actcccaaat agaactgagg 2040
atcctcgccc atctcagtgg tgatgagaat cttttgaggg cattcgaaga gggcatcgac 2100
gtccacactc taacagcttc cagaatattc aacgtgaaac ccgaagaagt aaccgaagaa 2160
atgcgccgcg ctggtaaaat ggttaatttt tccatcatat acggtgtaac accttacggt 2220
ctgtctgtga ggcttggagt acctgtgaaa gaagcagaaa agatgatcgt caactacttc 2280
gtcctctacc caaaggtgcg cgattacatt cagagggtcg tatcggaagc gaaagaaaaa 2340
ggctatgtta gaacgctgtt tggaagaaaa agagacatac cacagctcat ggcccgggac 2400
aggaacacac aggctgaagg agaacgaatt gccataaaca ctcccataca gggtacagca 2460
gcggatataa taaagctggc tatgatagaa atagacaggg aactgaaaga aagaaaaatg 2520
agatcgaaga tgatcataca ggtccacgac gaactggttt ttgaagtgcc caatgaggaa 2580
aaggacgcgc tcgtcgagct ggtgaaagac agaatgacga atgtggtaaa gctttcagtg 2640
ccgctcgaag tggatgtaac catcggcaaa acatggtcgt ga 2682
<210>22
<211>8
<212>PRT
＜213〉artificial
<220>
＜223〉the conservative motif A in dna polymerase activity site
<400>22
Asp Tyr Ser Gln Ile Glu Leu Arg
1 5
<210>23
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA exemplary unmodified reference of hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone in Polymerase Structure territory
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(10)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Gln, Thr, Met, Gly or Leu
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(19)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(20)
＜223〉Xaa=Glu or Asp
<220>
<221>MOD_RES
<222>(50)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(51)
＜223〉Xaa=Asp, Glu or Asn
<220>
<221>MOD_RES
<222>(76)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(77)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(78)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(79)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(80)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(81)
＜223〉Xaa=Ser, Ala, Val or Gly
<220>
<221>MOD_RES
<222>(82)
＜223〉Xaa=Ala or Leu
<400>23
Ile Val Glu Lys Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa
1 5 10 15
Thr Tyr Xaa Xaa Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Xaa Xaa Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>24
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉the exemplary unmodified of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6 is with reference to the Polymerase Structure territory
The avtive spot zone
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(10)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Gln, Thr, Met, Gly or Leu
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(19)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(20)
＜223〉Xaa=Glu or Asp
<220>
<221>MOD_RES
<222>(50)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(51)
＜223〉Xaa=Asp, Glu or Asn
<220>
<221>MOD_RES
<222>(77)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(78)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(79)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(80)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(81)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(82)
＜223〉Xaa=Ser, Ala, Val or Gly
<220>
<221>MOD_RES
<222>(83)
＜223〉Xaa=Ala or Leu
<400>24
Ile Ile Pro Leu Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa
1 5 10 15
Thr Tyr Xaa Xaa Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Xaa Xaa Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>25
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA exemplary unmodified reference of hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone in Polymerase Structure territory
<220>
<221>MOD_RES
<222>(50)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(51)
＜223〉Xaa=Asp, Glu or Asn
<400>25
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Asn Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Xaa Xaa Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>26
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉the exemplary unmodified of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6 is with reference to the Polymerase Structure territory
The avtive spot zone
<220>
<221>MOD_RES
<222>(50)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(51)
＜223〉Xaa=Asp, Glu or Asn
<400>26
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Xaa Xaa Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>27
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif a
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=D-or L-Ala, Cys, Asp, Glu, Phe, His, Ile, Lys,
Asn, Pro, Arg, Ser, Val, Trp or Tyr
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Glu or Asp
<400>27
Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa Thr Tyr Xaa Xaa
1 5 10 15
<210>28
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif a
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Arg, Lys or Asn
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Glu or Asp
<400>28
Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa Thr Tyr Xaa Xaa
1 5 10 15
<210>29
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif a
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Glu or Asp
<400>29
Xaa Xaa Xaa Xaa Arg Xaa Xaa Arg Lys Leu Xaa Xaa Thr Tyr Xaa Xaa
1 5 10 15
<210>30
<211>13
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif b
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=D-or L-Ala, Cys, Phe, Gly, His, Ile, Lys, Leu,
Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
<400>30
Thr Gly Arg Leu Ser Ser Xaa Xaa Pro Asn Leu Gln Asn
1 5 10
<210>31
<211>13
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif b
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Gly, Ala, Ser, Thr, Arg, Lys, Gln, Leu, Val or Ile
<400>31
Thr Gly Arg Leu Ser Ser Xaa Xaa Pro Asn Leu Gln Asn
1 5 10
<210>32
<211>13
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif b
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Gly, Thr, Arg, Lys or Leu
<400>32
Thr Gly Arg Leu Ser Ser Xaa Xaa Pro Asn Leu Gln Asn
1 5 10
<210>33
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=D-or L-Ala, Cys, Asp, Glu, Phe, Gly, His, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Ser, Ala, any amino acid outside Val or the Gly
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>33
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>34
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Phe or Tyr
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Ser, Ala, any amino acid outside Val or the Gly
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>34
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>35
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Ser, Ala, any amino acid outside Val or the Gly
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>35
Xaa Xaa Xaa Phe Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>36
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉any amino acid outside Xaa=Ile or the Leu
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
<223>Xaa＝Cys，Asp，Glu，Phe，His，Ile，Lys，Leu，Met，Asn，
Pro, Gln, Arg, Thr, Trp or Tyr
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>36
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>37
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉any amino acid outside Xaa=Ile or the Leu
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Phe or Tyr
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>37
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>38
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement archaeal dna polymerase is modified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉any amino acid outside Xaa=Ile or the Leu
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>38
Xaa Xaa Xaa Xaa Xaa Phe Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>39
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>39
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>40
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>40
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>41
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>41
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>42
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>42
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>43
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>43
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>44
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>44
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>45
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>45
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>46
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>46
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>47
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>47
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>48
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>48
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>49
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>49
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>50
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>50
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>51
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>51
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>52
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>52
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>53
<211>91
<212>PRT
＜213〉artificial
<220>
＜223〉the improvement sudden change polysaccharase of the archaeal dna polymerase that relies on from the thermally-stabilised family Aform DNA of the hot bacterial classification Z05 (Z05) of dwelling
The avtive spot zone of structural domain
<400>53
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Arg Lys Leu Lys Asn
1 5 10 15
Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly Arg
20 25 30
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu Gly
50 55 60
Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Phe Val
65 70 75 80
Phe Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
85 90
<210>54
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>54
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>55
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>55
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>56
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>56
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>57
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>57
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>58
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>58
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>59
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>59
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>60
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>60
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>61
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>61
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>62
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>62
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>63
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>63
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>64
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>64
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>65
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>65
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>66
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>66
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>67
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>67
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>68
<211>92
<212>PRT
＜213〉artificial
<220>
＜223〉active sites in the improvement sudden change Polymerase Structure territory of archaeal dna polymerase CS5 that relies on from chimeric heat-stable DNA or CS6
The point zone
<400>68
Ile Ile Pro Leu Ile Leu Glu Tyr Arg Lys Ile Arg Lys Leu Lys Ser
1 5 10 15
Thr Tyr Ile Asp Ala Leu Pro Lys Met Val Asn Pro Lys Thr Gly Arg
20 25 30
Ile His Ala Ser Phe Asn Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser
35 40 45
Ser Ser Gly Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly
50 55 60
Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Phe
65 70 75 80
Val Phe Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile
85 90
<210>69
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉archaeal dna polymerase unmodified motif a
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Leu or Gln
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Gln, His or Glu
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Tyr, His or Phe
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Glu, Gln or Lys
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ile, Leu or Tyr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Gln, Thr, Met, Gly or Leu
<220>
<221>MOD_RES
<222>(11)
＜223〉Xaa=Lys or Gln
<220>
<221>MOD_RES
<222>(12)
＜223〉Xaa=Ser or Asn
<220>
<221>MOD_RES
<222>(15)
＜223〉Xaa=Ile or Val
<220>
<221>MOD_RES
<222>(16)
＜223〉Xaa=Glu or Asp
<400>69
Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Lys Leu Xaa Xaa Thr Tyr Xaa Xaa
1 5 10 15
<210>70
<211>13
<212>PRT
＜213〉artificial
<220>
＜223〉archaeal dna polymerase unmodified motif b
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ser or Thr
<220>
<221>MOD_RES
<222>(8)
＜223〉Xaa=Asp, Glu or Asn
<400>70
Thr Gly Arg Leu Ser Ser Xaa Xaa Pro Asn Leu Gln Asn
1 5 10
<210>71
<211>15
<212>PRT
＜213〉artificial
<220>
＜223〉archaeal dna polymerase unmodified motif c
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=Gly, Asn or Asp
<220>
<221>MOD_RES
<222>(2)
＜223〉Xaa=Trp or His
<220>
<221>MOD_RES
<222>(3)
＜223〉Xaa=Trp, Ala, Leu or Val
<220>
<221>MOD_RES
<222>(4)
＜223〉Xaa=Ile or Leu
<220>
<221>MOD_RES
<222>(5)
＜223〉Xaa=Val, Phe or Leu
<220>
<221>MOD_RES
<222>(6)
＜223〉Xaa=Ser, Ala, Val or Gly
<220>
<221>MOD_RES
<222>(7)
＜223〉Xaa=Ala or Leu
<400>71
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Ser Gln Ile Glu Leu Arg
1 5 10 15
<210>72
<211>30
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic M13mp18 single stranded DNA templa-primer
<400>72
gggaagggcg atcggtgcgg gcctcttcgc 30
<210>73
<211>27
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic Oligonucleolide primers QX
<220>
＜221〉modify _ base
<222>(15)
＜223〉n=is by a of quencher dyestuff (Q) Black Hole quencher (BHQ) modification
<400>73
gcaagcaccc tatcnggcag taccaca 27
<210>74
<211>28
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic complementary oligonucleotide R1
<220>
<221>modified_base
<222>(28)
＜223〉n=is by the c of 3 ' phosphoric acid modification
<400>74
ttgtggtact gcctgatagg gtgcttgn 28
<210>75
<211>27
<212>DNA
＜213〉artificial
<220>
＜223〉the fluorescein-labeled primers F AM-QX of synthetic
<220>
＜221〉modify _ base
<222>(15)
＜223〉n=is by a of quencher dyestuff (Q) Black Hole quencher (BHQ) modification
<220>
＜221〉modify _ base
<222>(27)
＜223〉the fluorescein-labeled a that modified by 2 ' phosphoric acid of n=,
Fluorescein-labeled Desoxyadenosine four phosphoric acid (dA4P),
Fluorescein-labeled 2 '-terminating nucleotide
<400>75
gcaagcaccc tatcnggcag taccacn 27
<210>76
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉synthetic primer HC2
<400>76
gcagaaagcg tctagccatg gctta 25
Claims (according to the modification of the 19th of treaty)
1. archaeal dna polymerase, it comprises following at least motif in the Polymerase Structure territory:
T-G-R-L-S-S-X B7-X B8-P-N-L-Q-N (SEQ ID NO:32), wherein X B7Be S or T; And X B8Be to be selected from G, T, R, the amino acid of K or L,
Wherein said polysaccharase comprises 3 '-5 ' exonuclease activity, and with respect to X B8Be to be selected from D, the amino acid of E or N and the identical archaeal dna polymerase in other aspects have the reverse transcription efficient that improved nucleic acids acids is extended speed and/or improvement.
2. the archaeal dna polymerase of claim 1, wherein X B8Be the amino acid that is selected from G or K.
3. the archaeal dna polymerase of claim 1, wherein site X B8Amino acid be G.
4. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase has at least 90% sequence identity with the polysaccharase that is selected from the group of being made up of following polysaccharase:
(a) CS5 archaeal dna polymerase;
(b) CS6 archaeal dna polymerase;
(c) Thermotoga maritima archaeal dna polymerase;
(d) thermus aquaticus archaeal dna polymerase;
(e) thermus thermophilus archaeal dna polymerase;
(f) the Huang hot bacterium archaeal dna polymerase of dwelling;
(g) the thread hot bacterium archaeal dna polymerase of dwelling;
(h) the hot bacterial classification sps17 archaeal dna polymerase of dwelling;
(i) the hot bacterial classification Z05 archaeal dna polymerase of dwelling;
(j) Naples thermobacillus archaeal dna polymerase of dwelling;
(k) the Africa hot chamber bacterium archaeal dna polymerase of dwelling;
(1) Thermus caldophilus archaeal dna polymerase.
5. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase is CS5DNA polysaccharase or CS6 archaeal dna polymerase, and the 640th amino acids is selected from the group of being made up of G, T, R, K and L.
6. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase is the Z05DNA polysaccharase, and the 580th amino acids is selected from the group of being made up of G, T, R, K and L.
7. the archaeal dna polymerase of claim 1, wherein said polysaccharase comprises chimeric polysaccharase, and wherein said chimeric polysaccharase and the CS5 archaeal dna polymerase of SEQ ID NO:18 or the CS6 archaeal dna polymerase of SEQ ID NO:19 have at least 90% sequence identity.
8. the archaeal dna polymerase of claim 7, wherein said chimeric polysaccharase comprises the aminoacid replacement that one or more is selected from the group of being made up of following aminoacid replacement: G46E, L329A, Q601R, D640G, I669F, S671F and E678G with respect to SEQ ID NO:18 or SEQ ID NO:19.
9. the archaeal dna polymerase of claim 1, it further comprises hot reversible covalent modification.
10. the archaeal dna polymerase of claim 9, the wherein said polysaccharase that comprises hot reversible covalent modification by heat-stable DNA polymerase and the mixture of dicarboxylic anhydride with the general formula that is selected from the group of forming by following general formula at alkaline pH be lower than the reaction generation of carrying out under about 25 ℃ temperature:
(a) formula I:
Figure A20078003877201931
R wherein 1And R 2Be hydrogen or organic group, they can connect together; With
(b) formula II:
R wherein 1And R 2Be organic group, they can connect together, and hydrogen is cis.
11. coding is according to the recombinant nucleic acid of the archaeal dna polymerase of claim 1.
12. comprise the expression vector of the recombinant nucleic acid of claim 11.
13. comprise the host cell of the expression vector of claim 12.
14. a method of producing archaeal dna polymerase, described method comprises: the host cell of cultivating claim 13 under the condition of the expression of nucleic acid that is fit to the coding DNA polysaccharase.
15. a method of implementing primer extension comprises:
Under the condition that is fit to primer extension, the archaeal dna polymerase of claim 1 is contacted with primer, polynucleotide template and free nucleotide, thereby produce the primer that extends.
16. the method for claim 15, wherein said polynucleotide template is RNA.
17. the method for claim 16, the condition of wherein said suitable extension comprises Mg ++And Mn ++
18. according to the method for claim 15, wherein said free nucleotide comprises unconventional Nucleotide.
19. the method for claim 15, it is the method that is used for polynucleotide amplification, is included in archaeal dna polymerase and primer are contacted to, polynucleotide template and free nucleotide.
20. the method for claim 19, wherein said polynucleotide template is RNA.
21. the method for claim 20, the condition of wherein said suitable amplification comprises Mg ++And Mn ++
22. a test kit of producing the primer of extension comprises:
At least one provides the container according to the archaeal dna polymerase of claim 1.
23. the test kit of claim 22 comprises that further one or more is selected from the other container of the group of being made up of following container:
(a) be provided under the primer extension condition can with the container of the primer of predetermined polynucleotide template hybridization;
(b) provide the container of free nucleotide; With
(c) provide the container of the buffer reagent that is fit to primer extension.
24. a reaction mixture comprises the archaeal dna polymerase according to claim 1, at least a primer, polynucleotide template and free nucleotide.
25. the reaction mixture of claim 24, wherein said polynucleotide template is DNA.
26. the reaction mixture of claim 24, wherein said polynucleotide template is RNA.

Claims (28)

1. archaeal dna polymerase, it comprises the motif that at least one is selected from the group of being made up of following motif in the Polymerase Structure territory:
A) X A1-X A2-X A3-X A4-R-X A6-X A7-X A8-K-L-X A11-X A12-T-Y-X A15-X A16(SEQID NO:1); X wherein A1Be I or L; X A2Be L or Q; X A3Be Q, H or E; X A4Be Y, H or F; X A6Be E, Q or K; X A7Be I, L or Y; X A8Be other amino acid except that Q, T, M, G or L; X A11Be K or Q; X A12Be S or N; X A15Be I or V; And X A16Be E or D;
B) T-G-R-L-S-S-X B7-X B8-P-N-L-Q-N (SEQ ID NO:2); X wherein B7Be S or T; And X B8Be other amino acid except that D, E or N; With
C) X C1-X C2-X C3-X C4-X C5-X C6-X C7-D-Y-S-Q-I-E-L-R (SEQ ID NO:3); X wherein C1Be G, N, or D; X C2Be W or H; X C3Be W, A, L or V; X C4Be other amino acid except that I or L; X C5Be V, F or L; X C6Be other amino acid except that S, A, V or G; And X C7Be A or L,
Wherein this polysaccharase is with respect to X wherein A8Be the amino acid that is selected from Q, T, M, G or L; X B8Be the amino acid that is selected from D, E or N; And X C1Be the amino acid that is selected from S, A, V or G, and other identical archaeal dna polymerase have the reverse transcription efficient that improved nucleic acids acids is extended speed and/or improvement.
2. the archaeal dna polymerase of claim 1, wherein said polysaccharase comprises motif b) and c), and X B8Be other amino acid except that D, E or N, and X C6Be other amino acid except that S, A, V or G.
3. the archaeal dna polymerase of claim 1, wherein said polysaccharase comprise motif a), b) and c), and
Site X A8Amino acid be R;
Site X B8Amino acid be G;
Site X C4Amino acid be F; And
Site X C6Amino acid be F.
4. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase has at least 90% sequence identity with the polysaccharase that is selected from the group of being made up of following polysaccharase:
(a) CS5 archaeal dna polymerase;
(b) CS6 archaeal dna polymerase;
(c) Thermotoga maritima archaeal dna polymerase;
(d) thermus aquaticus archaeal dna polymerase;
(e) thermus thermophilus archaeal dna polymerase;
(f) the Huang hot bacterium archaeal dna polymerase of dwelling;
(g) the thread hot bacterium archaeal dna polymerase of dwelling;
(h) the hot bacterial classification sps17 archaeal dna polymerase of dwelling;
(i) the hot bacterial classification Z05 archaeal dna polymerase of dwelling;
(j) Naples thermobacillus archaeal dna polymerase of dwelling;
(k) the Africa hot chamber bacterium archaeal dna polymerase of dwelling;
(l) Thermus caldophilus archaeal dna polymerase.
5. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase is CS5 archaeal dna polymerase or CS6 archaeal dna polymerase, and the 640th amino acids is selected from the group of being made up of G, T, R, K and L.
6. the archaeal dna polymerase of claim 1, wherein said archaeal dna polymerase is the Z05 archaeal dna polymerase, and the 580th amino acids is selected from the group of being made up of G, T, R, K and L.
7. the archaeal dna polymerase of claim 1, wherein said polysaccharase comprises chimeric polysaccharase.
8. the archaeal dna polymerase of claim 7, wherein said chimeric polysaccharase and the CS5 archaeal dna polymerase of SEQ IDNO:18 or the CS6 archaeal dna polymerase of SEQ ID NO:19 have at least 90% sequence identity.
9. the archaeal dna polymerase of claim 8, wherein said chimeric polysaccharase comprises the aminoacid replacement that one or more is selected from the group of being made up of following aminoacid replacement: G46E, L329A, Q601R, D640G, I669F, S671F and E678G with respect to SEQID NO:18 or SEQ ID NO:19.
10. the archaeal dna polymerase of claim 1, it further comprises hot reversible covalent modification.
11. the archaeal dna polymerase of claim 10, the wherein said polysaccharase that comprises hot reversible covalent modification by heat-stable DNA polymerase and the mixture of dicarboxylic anhydride with the general formula that is selected from the group of forming by following general formula at alkaline pH be lower than the reaction generation of carrying out under about 25 ℃ temperature:
(a) formula I:
Figure A2007800387720003C1
R wherein 1And R 2Be hydrogen or organic group, they can connect together; With
(b) formula II:
R wherein 1And R 2Be organic group, they can connect together, and hydrogen is cis.
12. coding is according to the recombinant nucleic acid of the archaeal dna polymerase of claim 1.
13. comprise the expression vector of the recombinant nucleic acid of claim 12.
14. comprise the host cell of the expression vector of claim 13.
15. a method of producing archaeal dna polymerase, described method comprises: the host cell of cultivating claim 14 under the condition of the expression of nucleic acid that is fit to the coding DNA polysaccharase.
16. a method of implementing primer extension comprises:
Under the condition that is fit to primer extension, the archaeal dna polymerase of claim 1 is contacted with primer, polynucleotide template and free nucleotide, thereby produce the primer that extends.
17. the method for claim 16, wherein said polynucleotide template is RNA.
18. the method for claim 17, the condition of wherein said suitable extension comprises Mg ++And Mn ++
19. according to the method for claim 16, wherein said free nucleotide comprises unconventional Nucleotide.
20. the method for claim 16, it is the method that is used for polynucleotide amplification, is included in archaeal dna polymerase and primer are contacted to, polynucleotide template and free nucleotide.
21. the method for claim 20, wherein said polynucleotide template is RNA.
22. the method for claim 21, the condition of wherein said suitable amplification comprises Mg ++And Mn ++
23. a test kit of producing the primer of extension comprises:
At least one provides the container according to the archaeal dna polymerase of claim 1.
24. the test kit of claim 23 comprises that further one or more is selected from the other container of the group of being made up of following container:
(a) be provided under the primer extension condition can with the container of the primer of predetermined polynucleotide template hybridization;
(b) provide the container of free nucleotide; With
(c) provide the container of the buffer reagent that is fit to primer extension.
25. a reaction mixture comprises the archaeal dna polymerase according to claim 1, at least a primer, polynucleotide template and free nucleotide.
26. the reaction mixture of claim 25, wherein said polynucleotide template is DNA.
27. the reaction mixture of claim 25, wherein said polynucleotide template is RNA.
28. the reaction mixture of claim 26, wherein said reaction mixture comprises Mg ++And Mn ++
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